The human A549 lung cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) at 37°C in a 5% CO₂ humidified tissue culture incubator. To determine the effects of THSG on A549 cell adhesion and invasion, cells were exposedto 0, 10, 25 and 50 µM THSG (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) for 1, 2 and 3 h at 37°C prior to cell adhesion and invasion assays. For all experiments, the concentration of FBS was reduced to 2% and cells were treated for the indicated time periods with stock solutions of THSG prepared using dimethyl sulfoxide.

The viability of A549 cells was assessed using the CCK8 assay (Beyotime Institute of Biotechnology, Haimen, China). Briefly, A549 cells were seeded in 96-well plates at a density of 2×10³ cells/well with 100 ml complete culture medium. After incubating cells under standard conditions for 24 h, THSG was added to the medium at a final concentration of 0, 5, 10, 25, 50, 100, 150 and 200 µM. Cells were subsequently incubated for a further 12, 24 and 48 h at 37°C. CCK8 solution (20 µl) was then added to each well and cells were incubated for 1 h at 37°C. The optical density (OD) was read at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Cells growing in logarithmic phase were trypsinized using 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) and were then resuspended in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) medium containing 10% FBS. Cells were then seeded in a 12-well microplate at a density of 1×10⁵ cells/ml before they were incubated for 1, 2 and 3 h with different concentrations of THSG (0, 10, 25 and 50 µM) at 37°C. The supernatant was discarded and cells were washed twice with phosphate-buffered saline (Gibco; Thermo Fisher Scientific, Inc.). Paraformaldehyde (4%; JRDun Biotechnology Co., Ltd., Shanghai, China) was used to fix cells for 15 min, before they were stained with Giemsa (Beijing Solarbio Technology Co., Ltd., Beijing, China) for 30 min. The cells were then washed 3 times with PBS (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) and the OD was read at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The following formula was used to quantify cell adhesion: Adhesion rate (%)=(OD₁/OD₀)x100, where OD₁ indicates THSG-treated groups and OD₀ indicates the control group.

The cell invasion assay was performed using a 24-well Transwell chamber with an 8-µm pore size (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The inserts were coated with 50 µl Matrigel matrix (DMEM dilution, 1:2; BD Biosciences, Franklin Lakes, NJ, USA). Cells were trypsinized following treatment with various concentrations of THSG (0, 10, 25 and 50 µM) for 24 h at 37°C, and 1×10⁵ cells were transferred to the upper Matrigel chamber containing 100 µl of serum-free medium and incubated for a further 24 h. The lower chamber was filled with medium containing 10% FBS as a chemoattractant. Following incubation, the cells that had traversed the filter membrane were fixed and stained using 0.1% crystal violet. Cells were visualized under an OLYMPUS microscope (Olympus Corporation, Tokyo, Japan). For each sample, the number of invaded cells was counted in five high-power fields of view selected at random.

Cells were first seeded at a density of 5×10⁵ cells/well in 6-well plates, cultured overnight and were then treated with THSG (0, 10, 25 and 50 µM) for 24 h. The cultured cells (1×10⁶) were harvested, washed twice with PBS and lysed in ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) with 0.01% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min. The cell lysate was then centrifuged at 13,000 × g for 10 min at 4°C. The supernatant (30 µg/lane) was run on a 10% SDS-PAGE gel and transferred electrophoretically onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The blots were blocked with 5% skim milk, then incubated with antibodies against phosphorylated snail (cat. no. ab53519; 1:500; Abcam, Cambridge, MA, USA), E-cadherin (cat. no. ab133597; 1:1,000; Abcam), Vimentin (cat. no. ab137321; 1:1,000; Abcam), MMP-2 (cat. no. ab37150; 1:200; Abcam), MMP-9 (cat. no. ab137867; 1:1,000; Abcam) and GAPDH (cat. no. ab9485; 1:2,500; Abcam) at 4°C overnight. The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology) or HRP-conjugated anti-rabbit secondary antibody (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 1 h. Bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) and quantified with ImageJ software (v1.48u; National Institutes of Health, Bethesda, MD, USA); a total of 3 experimental repeats were performed.

Human lung cancer A549 cells were seeded at a density of 5×10⁵ cells/well in 6-well plates, cultured overnight and were then treated with THSG (0, 10, 25 and 50 µM) for 12 h at 37°C. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (2 µg) was reverse transcribed using the First Strand cDNA Synthesis kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's instructions. PCR amplification was performed for 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec, and a final extension at 60°C for 4 mininan ABI 7300 Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.), using SYBR Premix Ex Taq (Takara Biotechnology, Co., Ltd., Dalian, China). Each reaction mixture had a final reaction volume of 20 µl, which was composed of 2 µl cDNA, 2 µl primers, 10 µl Premix Taq and 6 µl nuclease-free water. The specific primer sequences for each gene were as follows: E-cadherin, forward, 5′-GTTGTTGGGCATAGAGAC-3′, reverse, 5′-CAGGGCAGTTTGAATAGC-3′ (product, 125 bp); vimentin, forward, 5′-GCGTGAAATGGAAGAGAAC-3′, reverse, 5′-TGGAAGAGGCAGAGAAATC-3′ (product, 217 bp); Snail, forward, 5′-TTCCTGAGCTGGCCTGTCTG-3′, reverse, 5′-TGGCCTGAGGGTTCCTTGTG-3′ (product, 165 bp); MMP2, forward, 5′-TTGACGGTAAGGACGGACTC-3′, reverse, 5′-GGCGTTCCCATACTTCACAC-3′ (product, 134 bp); MMP9, forward, 5′-AAGGGCGTCGTGGTTCCAACTC-3′, reverse, 5′-AGCATTGCCGTCCTGGGTGTAG-3′ (product, 210 bp); and GAPDH, forward, 5′-CACCCACTCCTCCACCTTTG-3′ and reverse, 5′-CCACCACCCTGTTGCTGTAG-3′ (product, 110 bp). All primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Target gene expression levels were determined using the 2^−ΔΔCq method of relative quantification (14), and all samples were normalized to GAPDH expression, which was used as an endogenous control.

The GraphPad Prism software program (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was employed for performing statistical analysis of data. Data are expressed as the mean ± standard deviation. One-way analysis of variance followed by Dunnett's post hoc test were used for statistical analyses. All tests performed were two-sided. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1B, the in vitro effects of THSG on A549 cell viability were determined using the CCK8 assay. Compared with the untreated control group, 75, 100, 150 and 200 µM THSG significantly decreased A549 cell viability (P<0.05). Therefore, 10, 25 and 50 µM THSG were selected for use in subsequent experiments.

Adhesion of cancer cells to the extracellular matrix and basement membrane is considered to be an initial step in the invasive process of tumor metastasis (15,16). Therefore, the effects of various concentrations of THSG on human A549 lung cancer cell adhesion were investigated. As shown in Fig. 2, 10, 25 and 50 µM THSG significantly suppressed the adhesion of A549 cells at 1, 2 and 3 h, when compared with the untreated control group (P<0.01). These results suggest that THSG may inhibit lung cancer cell adhesion in a time and dose-dependent manner.

The invasive capabilities of human A549 lung cancer cells following treatment with THSG were determined using a Transwell assay. As shown in Fig. 3A and B, the invasiveness of cells treated with 10, 25 and 50 µM THSG were significantly decreased when compared with the control group in a concentration-dependent manner (P<0.05 and P<0.01). The invasion rates of cells treated with 10, 25 and 50 µM THSG were 71.25±14.21, 45.24±12.24 and 21.25±8.25%, respectively compared with the untreated control (100%).

E-cadherin is a tumor suppressor protein that is used as a prognostic marker for patients with esophageal cancer (17). Vimentin is an intermediate filament protein, which interacts with microtubule and actin microfilaments that constitute the body of the cytoskeleton (18). Therefore, E-cadherin and vimentin are reliable indicators for the assessment of cell adhesion. Snail is a transcription factor that regulates the expression of E-cadherin (19,20). RT-qPCR and western blot analyses were conducted to detect the mRNA and protein expression levels of Snail, E-cadherin and vimentin, respectively (Fig. 4). As shown in Fig. 4B, E-cadherin mRNA expression levels were significantly increased in a dose-dependent manner following treatment with 10, 25 and 50 µM THSG compared with the untreated control group (P<0.01). Conversely, vimentin and Snail mRNA expression levels were significantly decreased in a dose-dependent manner following treatment with 10, 25 and 50 µM THSG, as compared with the untreated control group (P<0.05 and P<0.01; Fig. 4A and C). Consistent with the mRNA expression levels, the protein expression levels of Snail and vimentin were significantly decreased, whereas E-cadherin protein expression was significantly increased following treatment with 10, 25 and 50 µM THSG compared with the untreated control group (P<0.05 and P<0.01; Fig. 4D and E).

Lung cancer cells produce MMPs, and an increase in the expression of these proteins has been associated with disease progression (21,22). In addition, the expression levels of MMP2 and MMP9 are significantly associated the invasive capabilities of cancer cells (23). To investigate the potential anti-invasive mechanisms of THSG in lung cancer cells in vitro, the expression levels of MMP2 and MMP9 in A549 cells exposed to various concentrations of THSG were detected. Western blot and RT-qPCR analyses were conducted to assess the expression levels of MMP2 and MMP9 in cells treated with 10, 25 and 50 µM THSG. As shown in Fig. 5, THSG exerted significant inhibitory effects on the mRNA and protein expression levels of MMP2 and MMP9 in a dose-dependent manner, as compared with the untreated control group (P<0.05; Fig. 5).