DMEM (Gibco; Thermo Fisher Scientific, Inc) with 10% FBS (Gemini Bio-Products, Inc.), 10 U/ml penicillin-G and 10 mg/ml streptomycin (Gemini Bio-Products, Inc.) was used to culture the CSCC cell line SCC13 (kind gift from Dr James Rheinwald, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA) at 37°C with 5% CO₂. SCC13 cells were treated with different concentrations of AL (0, 10, 30, 100 or 300 µM; purity >99%; Chengdu Best-Reagent Chemical, Co., Ltd.) (17) at 37°C for 24, 48 and 72 h (18) prior to subsequent experimentation.

A CCK-8 assay (Dojindo Molecular Technologies, Inc.) was used to measure cell viability according to the manufacturer's protocol. SCC13 cells were plated into 96-well plates at 5×10³ cells/well and incubated at 37°C for 24 h. Subsequently, the cells were treated with various concentrations (0, 10, 30, 100 or 300 µM) of AL for 24, 48 and 72 h at 37°C. Then, 10 µl CCK-8 assay solution was added to each well (Dojindo Molecular Technologies, Inc.) and the wells were incubated at 37°C for a 1 h. A microplate reader (Synergy2; BioTeK Instruments, Inc.) was used to measure the optical density at 450 nm. The IC₅₀ value of AL to SCC13 cells at 48 h was calculated. Each experiment was performed in triplicate.

To detect SCC13 cell apoptosis, the cells were treated with or without different concentrations of AL (10, 30, 100 or 300 µM) at 37°C for 48 h. Then, 5 µl Annexin V-FITC and 10 µl propidium iodide (cat. no. 70-AP101-100; Hangzhou Multi Sciences Biotech Co., Ltd.) were used to stain the cells at room temperature for 30 min following the manufacturer's protocol. Cell apoptosis was analyzed using a BD FACSCalibur™ flow cytometer (Becton, Dickinson and Company) and WinMDI software (version 2.5; Purdue University Cytometry Laboratories) was used to analyze the data.

SCC13 cells were treated with or without AL (10, 30, 100 or 300 µM) at 37°C for 48 h. Total cellular proteins from SCC13 cells were extracted using RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitor (Beyotime Institute of Biotechnology). A bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to evaluate the protein concentrations and 100°C water was used to heat the samples for 10 min to denature the proteins. Then, 12% SDS-PAGE gels were used to separate the proteins (40 µg per lane), which were then electrophoretically transferred to PVDF membranes (EMD Millipore). Membranes were blocked at room temperature for 1 h with 5% non-fat milk and blotted overnight at 4°C with the following primary antibodies (all from Cell Signaling Technology, Inc.): E-cadherin (1:1,000; cat no. 3195), N-cadherin (1:1,000; cat no. 13116), matrix metalloproteinase (MMP-9; 1:1,000; cat no. 13667), vimentin (1:1,000; cat no. 12826), Bcl-2 (1:1,000; cat no. 4223), Bax (1:1,000; cat no. 5023), phosphorylated (p)-mitogen-activated protein kinase kinase (p-MEK; 1:1,000; cat no. 3958), MEK (1:1,000; cat no. 8727), p-p65 (1:1,000; cat no. 3033), p65 (1:1,000; cat no. 8242) and β-actin (1:1,000; cat no. 4970). Membranes were then incubated with the anti-rabbit IgG horseradish peroxidase-linked secondary antibody (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 2.5 h at room temperature. Enhanced chemiluminescence reagent (EMD Millipore) was used to visualize the protein bands. The band density was quantified with Gel-Pro Analyzer densitometry software (version 6.3; Media Cybernetics, Inc.).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to prepare the total RNA from SCC13 cells according to the manufacturer's protocol. A PrimeScript™ RT reagent kit (Takara Bio, Inc.) was used to perform the RT. The temperature protocol for the reverse transcription reaction was as follows: Primer annealing at 25°C for 5 min, cDNA synthesis at 42°C for 60 min and termination at 80°C for 2 min. qPCR analysis was carried out using SYBR Premix Ex Taq II (Takara Bio, Inc.) following the manufacturer's instructions. The reaction conditions for PCR were: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C, 72°C for 30 sec and 78°C for 1.5 min. GAPDH was used as an internal control. All PCR primer sequences were obtained as required and listed as follows: E-cadherin forward, 5′-CGAGAGCTACACGTTCACGG-3′ and reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′; N-cadherin forward, 5′-TTTGATGGAGGTCTCCTAACACC-3′ and reverse, 5′-ACGTTTAACACGTTGGAAATGTG-3′; MMP-9 forward, 5′-AGACCTGGGCAGATTCCAAAC3′ and reverse, 5′-CGGCAAGTCTTCCGAGTAGT-3′; vimentin forward, 5′-GACGCCATCAACACCGAGTT-3′ and reverse, 5′-CTTTGTCGTTGGTTAGCTGGT-3′; Bcl-2, forward 5′-TTGGATCAGGGAGTTGGAAG-3′ and reverse, 5′-TGTCCCTACCAACCAGAAGG-3′ and Bax forward, 5′-CGTCCACCAAGAAGCTGAGCG-3′ and reverse, 5′-CGTCCACCAAGAAGCTGAGCG-3′. The 2^−ΔΔCq quantification method (19) was used to analyze the relative gene expression.

Data are presented as the mean ± SD. SPSS 16.0 software (SPSS, Inc.) was used to perform the statistical analysis. One-way ANOVA test followed by Tukey's post hoc test was used to assess the differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The present study investigated the effect of AL on CSCC cell viability using a CCK-8 assay. The present results suggested that treatment with AL at different concentrations (10, 30, 100 or 300 µM) for 24, 48 and 72 h significantly inhibited cell viability in a dose- and time-dependent manner compared with control cells without AL treatment (Fig. 1). The present results suggested that AL had cytotoxic effects on SCC13 cells in a dose- and time-dependent manner. In addition, the present results indicated that the IC₅₀ for 48 h of AL in the SCC13 cells was 80.27 µM.

The present study investigated whether AL affected CSCC cell apoptosis. SCC13 cells were treated with different concentrations of AL (10, 30, 100 or 300 µM) for 48 h, and then cell apoptosis was analyzed. AL dose-dependently increased the apoptotic ratio of SCC13 cells (Fig. 2). Therefore, the present results suggested that AL increased CSCC cell apoptosis.

To investigate whether AL has a role in EMT regulation, the present study evaluated the expression levels of the EMT markers E-cadherin, N-cadherin, MMP-9 and vimentin. The present results suggested that AL increased E-cadherin protein expression level and decreased N-cadherin, MMP-9 and vimentin protein expression levels in SCC13 cells in a dose-dependent manner (Fig. 3A-E). Similar results for the mRNA expression levels were indicated by RT-qPCR analysis (Fig. 3F-I).

The present study investigated the molecular mechanisms by which AL may affect SCC13 cells. SCC13 cells were treated with different concentrations of AL for 48 h, and then the expression levels of Bcl-2 and Bax and the relative proteins in MEK/NF-κB signaling pathway were measured. The present results suggested that Bcl-2, which plays an important role in apoptosis (20), was decreased in SCC13 cells after AL treatment (Fig. 4A and B). In addition, Bax protein expression level was significantly increased by AL treatment in a dose-dependent manner (Fig. 4A and C). A similar result was identified at the mRNA expression level of Bcl-2 and Bax (Fig. 4D and E). Moreover, the expression levels of the MEK/NF-κB signaling-related proteins p-MEK and p-p65 were significantly decreased (Fig. 5A-C), while AL treatment had no significant effect on the mRNA expression levels of MEK and p65 in SCC13 cells (Fig. 5D and E).