TCS was purchased from the Jinshan Pharmaceutical Co, Ltd. (Shanghai, China). Cisplatin was obtained from the Jingang Pharmaceutical Co Ltd. (Nanjing, China). The HEp-2 and AMC-HN-8 human laryngeal epidermoid carcinoma cell lines were provided by the Cell Bank of the Chinese Institute of Biochemistry and Cell Biology (Shanghai, China). The cell counting kit-8 (CCK-8) assay and Hochest33258 were purchased from Dojindo (Kumamoto, Japan). The JNK inhibitor, SP600125, was purchased from Sigma-Aldrich (St. Louis, MO, USA). The CytoTox 96 non-radioactive cytotoxicity assay was obtained from Promega Corportation (Madison, WI, USA). Polyclonal rabbit anti-human antibodies against caspase-3, caspase-9, JNK, phosphorylated (phospho)-JNK (Thr183/Tyr185), phospho-extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204) and phospho-p38 (Thr180/Tyr182) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

All the cells were maintained in RPMI-1640 medium, which was purchased from Gibco-BRL (Grand island, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL), penicillin (100 units/ml; Beyotime, Jiangsu, China) and streptomycin (100 μg/ml; Beyotime) in a 5% CO₂ incubator at 37°C. For the present study, the cells were incubated in either the presence or absence of different concentrations of TCS (1, 5, 9 or 13 μg/ml), with or without 3 μg/ml cisplatin for 5 days.

The inhibitory effects of the various treatments on HEp-2 and AMC-HN-8 cell viability were determined by CCK-8 assay. The cells, in the exponential growth phase, were seeded into 96-well plates with 100 μl (1×10⁴/ml) per well. Subsequently, different concentrations of TCS (1, 5, 9 and 13 μg/ml) were added. Each treatment was performed in triplicate wells and a control group consisting of cells grown in culture medium containing no drugs was included. The plates were placed in an incubator in a humidified atmosphere at 37°C for 5 days. At the end of the exposure, 10 μl CCK-8 was added to each well and the plates were incubated at 37°C for 1 h. The absorbance of each well was then measured using a standard enzyme-linked immunosorbant assay at 450 and 650 nm and the absorbance values were labeled A450 and A650, respectively. The inhibitory rate was calculated as follows: HEp-2 or AMC-HN-8 cells (100%) = 1 − (Atreat490 − Atreat650)]/(Acontrol490 − Acontrol650) ×100% and the results were measured in triplicate. SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to calculate the 50% inhibitory concentration for each group of drugs and the cell proliferation was analyzed by counting the number of cells with a hemocytometer (Hausser Scientific, Horsham, PA, USA) using trypan blue (Sangon Biotech, Shanghai, China) exclusion as the criterion for cell viability.

Following treatment of the HEp-2 and AMC-HN-8 cells with different concentrations of TCS (final concentrations of 1, 5, 9 and 13 μg/ml) for 48 h, the culture supernatant was harvested by centrifugation at 670 × g for 10 min. The supernatant (50 μl) from each well of the assay was transferred to the corresponding well of a flat-bottomed 96-well enzymatic assay plate with 50 μl reconstituted substrate mix (CytoTox 96^® non-radioactive cytotoxicity assay; Promega Corporation, Madison, WI, USA). The assay plate was then incubated at room temperature and protected from light for 30 min. The absorbance of each well was then measured at 490 nm following the addition of 50 μl stop solution (Promega Corportation).

The cells were cultured on cover slides and incubated in the presence of 5 μg/ml TCS for 48 h. Following fixation with 1% glutaraldehyde (Sangon Biotech) for 30 min, the cells were stained with 10 μg/ml Hoechst 33258 at room temperature for 10 min. For each cover slide, 1,500 cells were observed under a Leica fluorescence microscope (magnification, ×200; model, Laser 355,375, Leica Microsystems, Wetzlar, Germany) and images were captured using a digital camera (Leica Microsystems). Apoptosis, a form of cell death, is characterized by morphological changes, including chromatin condensation and apoptotic body formation. The percentage of cells undergoing apoptosis was determined (15).

The cells were treated either with different concentrations of TCS or 3 μg/ml cisplatin for 48 h. The cells were then trypsinized, harvested by centrifugation at 536 × g for 5 min, washed twice and resuspended in 100 μl binding buffer (eBioscience, San Diego, CA, USA). The cell density was adjusted to 1×10⁵/ml and the cells were incubated with 5 μl annexin V-fluorescein isothiocyanate (FITC) and 5 μl PI for 15 min at room temperature. The samples were analyzed using a fluorescence-activated cell sorting (FACS)scan flow cytometer (Becton-Dickinson, New York, NY, USA). Early apoptosis and necrosis were determined as the percentage of annexin V positive/PI negative or the percentage of annexin V positive/PI positive cells.

The present study synchronized the cells in the G0-phase through serum deprivation prior to treatment. After 3 days culture in medium containing 5 μg/ml TCS, the HEp-2 and AMC-HN-8 cells were collected by trypsinization, washed with phosphate-buffered saline (PBS; Sangon Biotech, Shanghai, China) and fixed in ice-cold 70% ethanol for 24 h. The cells were then stained with 1 ml PI (0.1 mg/ml with 0.1% Triton X-100; Sangon Biotech) and incubated in the dark for 30 min. The samples were analyzed by flow cytometric analysis using FACSCalibur (Becton-Dickinson). PI is a highly water soluble, fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. PI binds to DNA by intercalating between the bases in double-stranded nucleic acids in exposed nuclei (16).

The total RNA was extracted from the HEp-2 and AMC-HN-8 cells using aguanidium thiocyanate hot phenol-chloroform method (17). This total RNA (100 ng) was used for the synthesis of first-strand cDNA by RT in a reaction mixture (20 μl) containing 5 μM random hexamers, 2.5 μM oligo (dT) primer and 0.1 μM specific primer (Takara Bio, Inc., Shiga, Japan). The reaction was performed at 37 °C for 15 min and at 85°C for 5 sec.

The RT-qPCR reactions were performed using an ABI PRISM 7500 RT-qPCR system (Applied Biosystems, Foster City, CA, USA). For data analysis, the 2^−ΔCT (18) method was used to calculate the fold change. GADPH was considered to be unaffected by the treatment conditions in the present study and was used as a reference gene for normalization of the threshold cycle. The following primer sequences for the genes were used: p21, forward 5′-TGTCCGTCAGAACCCATGC-3′ and reverse 5′-AAAGTCGAAGTTCCATCGCTC-3′; p27, forward 5′-ATCACAAACCCCTAGAGGGCA-3′ and reverse 5′-GGGTCTGTAGTAGAACTCGGG-3′; Ki67, forward 5′-AGAAGAAGTGGTGCTTCGGAA-3′ and reverse 5′-AGTTTGCGTGGCCTGTACTAA-3′; p53, forward 5′-ACAGCTTTGAGGTGCGTGTTT-3′ and reverse 5′-CCCTTTCTTGCGGAGATTCTCT-3′ and PCNA, forward 5′-ACACTAAGGGCCGAAGATAACG-3′ and reverse 5′-CAGCATCTCCAATATGGCTGAG-3′. The following conditions were used: 1 cycle at 95°C for 30 sec, 40 cycles as 95°C for 5 sec, 34 seconds at 60°C, 1 cycle at 95° for 15 sec, 60 sec at 60°C and 15 sec at 95°C.

The whole cell extracts were prepared by directly dissolving the cells in 1X SDS loading buffer (Beyotime Institute of Biotechnology, Shanghai, China). Equal quantities (1×10⁵ cells/lane) of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (15% SDS-polyacrylamide gel electrophoresis for caspase-9 and caspase-3), transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA, USA) and inhibited at room temperature for 30 min with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST; Sangon Biotech) containing 20 mm Tris-HCl (pH 7.6), 500 mm NaCl and 0.1% (v/v) Tween 20. The blots were incubated overnight at 4°C with the following primary antibodies diluted in TBST buffer: JNK, phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), caspase-9 or caspase-3 primary antibodies (1:5,000). Following washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:4,000; KPL, Gaithersburg, MD, USA) and were visualized using an enhanced chemiluminescence detection kit (Thermo Scientific, Inc., Fair Lawn, NJ, USA) according to the manufacturer’s instructions.

SPSS 16.0 software (SPSS, Inc.) was used for data analysis. All data are expressed as the mean ± standard deviation from at least two independent experiments. A two-sided Student’s t-test was used to compare continuous variables between two groups. P<0.05 was considered to indicate a statistically significant difference.

To validate the suppressive effect of TCS on cell viability, the HEp-2 and AMC-HN-8 cells were treated with a range of concentrations (0–13 μg/ml) of TCS for 5 days followed by CCK-8 assay analysis. TCS inhibited the viability of the HEp-2 and AMC-HN-8 cells in a dose-and time-dependent manner (Fig. 1A and B). According to the slope of growth inhibition, the most significant effects of TCS were observed after 2 days of treatment. Lactate dehydrogenase (LDH) is a stable cytosolic enzyme, which can be released during the necrosis process (19). However, no differences in LDH levels were observed in the culture supernatant between the day 2 experiment group and the control group (P>0.05), suggesting that the suppressive activity of TCS was independent of necrosis (Fig. 1C and D). LDH is released upon cell lysis; however, necrosis is characterized by cell swelling and rapid loss of membrane integrity, causing LDH to be released into the cell culture supernatant, which was assessed in the present study using a CytoTox 96^® non-radioactive cytotoxicity assay (20). During apoptosis, cells undergo nuclear and cytoplasmic shrinkage, chromatin is partitioned into multiple fragments and the cells are fragmented into multiple membrane-surrounded apoptotic bodies, however the integrity of the cell membrane is retained during this process. Therefore, TCS significantly suppresses the viability of HEp-2 and AMC-HN-8 cells through apoptosis or another method that is independent of necrosis (15).

The HEp-2 and AMC-HN-8 cells were treated with TCS (5 μg/ml), cisplatin (3 μg/ml) or the two combined for 2 days followed by staining with Hoechst 33258. The morphological features of apoptosis, including the primary feature of apoptotic body formation, were then visualized by fluorescence microscopy (Fig. 2A and B). The results revealed significantly more apoptotic cells in the combination group compared with either the TCS- or cisplatin-only group when the total number of apoptotic cells were counted on a 1 cm² cover slip (P<0.05, supplemental Fig. 1). The apoptotic HEp-2 and AMC-HN-8 cells were also examined following treatment with different concentrations of TCS (0–13 μg/ml) for 48 h using flow cytometry. The lower left quadrant of each panel in Fig. 2 indicated viable cells, which excluded PI and were negative for annexin V-FITC binding (Fig. 2C and D). The upper right quadrants indicated non-viable, necrotic cells, which were positive for annexin V-FITC binding and PI uptake (Fig. 2C and D). The percentage of annexin V-FITC positive/PI negative cells, which indicated cells with an exposed phospholipid and intact plasma membrane, increased between 1.53 and 13.4% in the HEp-2 group and between 3.14 and 22.4% in the AMC-HN-8 group in the presence of 0–13 μg/ml TCS for 48 h. Collectively, these data confirmed that TCS induced apoptosis in the HEp-2 and AMC-HN-8 human laryngeal epidermoid carcinoma cells in a dose-dependent manner. However, it remains to be elucidated whether 13 μg/ml TCS and 3 μg/ml cisplatin shared similar inhibitory effects on the HEp-2 cells (69.64±3.1% and 67.38±5.0%, respectively) and AMC-HN-8 cells (75.66±4% and 59.58±3.3%, respectively) on the second day (Fig. 1A and B). The cisplatin-induced apoptotic rate was markedly higher, suggesting that the increased suppressive effect of TCS on the HEp-2 and AMC-HN-8 cells was partly independent of apoptosis.

To investigate the potential suppressive effects of TCS on the HEp-2 and AMC-HN-8 cells not caused by apoptosis, the cell cycle was analyzed. Cell cycle analysis indicated that culture with TCS for 48 h induced cell cycle arrest, as revealed by changes in the percentages of cells in each cell cycle sub-phase (Fig. 3A and B). After 3 days, the majority of the TCS-treated HEp-2 and AMC-HN-8 cells exhibited a prolonged S-phase (Fig. 3A). On the second day, the percentage of cells in the S-phase was significantly increased and the percentage of cells in the G1-phase was significantly decreased compared with the control (P<0.01; Fig. 3B). Based on this observation, it was hypothesized that TCS may impair DNA replication in the HEp-2 and AMC-HN-8 cells or may activate cyclin-dependent kinase (CDK) inhibitors (CKIs) to arrest the cells in the early S-phase. Consistent with this hypothesis, the cell proliferation assay indicated that TCS treatment resulted in a significant reduction in the number of growing cells compared with the untreated cells (Fig. 3C). To further determine the molecular mechanism underlying the TCS-induced S-phase cell cycle arrest, RT-qPCR was performed. Cell cycle-regulatory genes, including p27^kip1 and p21^Wafl/^cip1, were examined by RT-qPCR in the cells pre-treated with 5 μg/ml TCS for 48 h. As expected, the mRNA expression levels of p27^kip1 and p21^Wafl/^cip1 were significantly upregulated following TCS stimulation for 48 h. The expression levels of the p21^Wafl/^cip1 family CKIs, p21^Wafl/^cip1 and p27^kip1, are important in the precise regulation of CDK activity, which is essential for normal cell cycle progression (21).

The activation of JNK, which occurs through phosphorylation at Thr183 and Tyr185, was investigated by western blot analysis of the HEp-2 and AMC-HN-8 cells following treatment with different concentrations of TCS for different durations. Compared with the control group, the increased concentrations of TCS and the increased treatment durations caused upregulation in the levels of 54/46 kDa phospho-JNK fragments (Fig. 4). However, no significant differences were observed in the protein levels of JNK between the different experimental groups. In these experiments, βactin was used as a control to confirm equal loading. JNK/MAPK is important in cell proliferation, differentiation and apoptosis and the present study hypothesized that the antitumor activity of TCS may be mediated by the activation of JNK/MAPK.

In general, TCS treatment reduced the viability of the HEp-2 and AMC-HN-8 cells, as discussed previously. Compared with HEp-2 cells treated with 5 μg/ml TCS alone, co-administration with SP600125 and TCS increased the cell viability between 52.2±9.3% and 63.58±3.5%, respectively, and suppressed the inhibitory effect of TCS on the HEp-2 cells. This result was also observed in the AMC-HN-8 cells. Increasing the concentration of SP600125 caused further increases in cell viability, however, the magnitude of the effect differed (Fig. 5A and B). In addition, SP600125 decreased the apoptotic rate (Fig. 5; upper right + lower right quadrants) of the HEp-2 cells between 13.56% and 4.67% and AMC-HN-8 cells between 21.8 and 5.67%. To investigate the involvement of various caspase pathways during TCS-induced apoptosis, western blot analysis of the TCS-treated cells was performed using specific antibodies against caspase-3 and caspase-9. The cells were treated with 5 μg/ml TCS and different concentrations of SP600125 (0–10 μM) for 48 h followed by protein extract preparation and SDS-PAGE fractionation. SP600125 is a known JNK inhibitor, which inhibits JNK/MAPK phosphorylation (22), however, SP600125 did not affect p38/MAPK or ERK/MAPK phosphorylation (Fig. 5D and E). Furthermore, the TCS-induced caspase-9 and caspase-3 cleavage was inhibited by SP600125 in a dose-dependent manner. These results suggested that JNK may be important in TCS-induced apoptosis and the inhibition of cell viability.