Ordn (chemical structure shown in Fig. 1A) was kindly provided by professor Zigang Dong of China-US (Henan) Hormel Cancer Institute (Zhengzhou, Henan, China). Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), trypsin, penicillin and streptomycin and phosphate-buffered saline (PBS) were purchased from HyClone (Logan, UT, USA). Antibodies against CHOP (sc-793), DR4 (sc-7863), DR5 (sc-166624), poly(ADP-Ribose) polymerase (PARP)-1 (sc-7150), p21 (sc-6246), p27 (sc-528), Mcl-1 (sc-819), survivin (sc-17779), Bax (sc-493), cyto c (sc-13156), α-tubulin (sc-5286), cytochrome c oxidase 4 (COX4; sc-69359), apoptotic protease activating factor-1 (Apaf-1; sc-33870) and actin (sc-1615) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The specific antibodies to JNK (#9252), p-JNK (Thr183/Tyr185; #9251), p38 (#9212), p-p38 (Thr180/Tyr182; #9211) and tBid (#2002) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Basal Medium Eagle, 4′-6-diamidino-2-phenylindole (DAPI), N-acetyl-L-cysteine (NAC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

The HN22 (RRID:CVCL_5522) cell line has been described previously (26), and was provided by Dankook University (Cheonan, Korea). The HSC4 (RRID:CVCL_1289) cell line was obtained from the Human Science Research Resources Bank (Osaka, Japan), and was provided by Hokkaido University (Hokkaido, Japan). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FBS and 100 U/ml each of penicillin and streptomycin at 37°C in a 5% CO₂ incubator.

The HN22 (2 × 10³/well) and HSC4 (2.5 × 10³/well) cells were seeded into 96-well plates. Following incubation overnight, the adherent cells were exposed to various concentrations (0, 5, 7.5 and 10 µM) of Ordn for 24 and 48 h. Following treatment, 30 µl of MTT solution (5 mg/ml) were added to each well followed by incubation for a further 2 h at 37°C. The supernatant was subsequently removed and DMSO was then added to the cells. To solubilize the formazan, the 96-well plates were gently mixed on a gyratory shaker for 5 min at 37°C. The absorbance of the formazan solution was recorded at a wavelength of 570 nm by Enspire Multimode Plate reader (Perkin-Elmer, Akron, OH, USA). The viability results are expressed as the IC₅₀ mean values of 3 independent experiments.

The oral cancer cells suspended in Basal Medium Eagle supplemented with FBS, gentamicin and L-glutamine were added to 0.3% agar in a top layer over a base layer of 0.6% agar containing Ordn (1, 2 and 4 µM). The cultures were maintained at 37°C in a 5% CO₂ incubator for 3 weeks, and then the cell colonies were counted under a microscope (Olympus Corporation, Tokyo, Japan).

The number of cells undergoing apoptosis was quantified after DAPI staining. Briefly, he HN22 and HSC4 cells were treated with Ordn (5, 7.5 and 10 µM) for 48 h and then harvested by trypsinization. The cells were washed a third time with PBS and centrifuged at 850 × g for 5 min at 4°C. The cell pellets were fixed in 100% methanol at room temperature for 30 min. The cells were deposited on slides and stained with DAPI solution in the dark. Subsequently, the DAPI-stained apoptotic cells was observed under an Olymps IX79-DP73 fluorescence microscope (Olympus Corporation).

The assay was performed using the Muse™ Cell Cycle kit (MCH100106; Merck Millipore, Billerica, MA, USA) to measure the DNA content at cell cycle stages (GO/G1, S and G2/M), as previously described (27). Either the HN22 or the HSC4 cells were plated in a 6-well plate and treated with Ordn at various concentrations (0, 5, 7.5 and 10 µM) for 48 h at 37°C. The cells were harvested and then suspended in cold PBS. The cell pellets were fixed in cold 70% ethanol for overnight at −20°C. After washing again with cold PBS, the cells were stained with Muse™ Cell Cycle kit reagent. Following 30 min of incubation at room temperature in the dark, the cell cycle distribution was analyzed using the Muse™ cell analyzer flow cytometer (Merck Millipore).

According to the manufacturer's instructions, the assay was carried out using the Muse™ Annexin V and Dead Cell kit (MCH100105; Merck Millipore). Briefly, the HN22 and HSC4 cells were seeded in a 6-well plate and incubated at 37°C for 24 h. The cells were treated with Ordn (0, 5, 7.5 and 10 µM), harvested, washed twice with cold PBS and transferred to 1.5 ml microcentrifuge tubes. Muse™ Annexin V and Dead Cell reagent was then added to each tube, followed by incubation for a further 20 min at room temperature in the dark. The analyses of apoptotic cells were carried out using the Muse™ cell analyzer.

The assay was performed using the Muse™ cell Analyzer to determine oxidative stress induced by Ordn. The Muse™ Oxidative Stress Kit (MCH100111; Merck Millipore) allows for the quantitative measurements of ROS levels in cells subjected to oxidative stress. Following treatment with Ordn (0, 5, 7.5 and 10 µM), the HN22 and HSC4 cells were collected. Following centrifugation (1,5 00 g, 5 min, room temperature), the cells were resuspended in 1X assay buffer. Finally, 190 µl of Muse™ Oxidative Stress working solution was mixed with 10 µl of the cell suspension and incubated at 37°C for 30 min prior to analysis. Following incubation, the stained cells were examined using the Muse™ cell analyzer.

To examine the changes in mitochondrial transmembrane potential at the early stages, MMP was measured using the Muse™ Cell Analyzer with the Muse MitoPotential Assay kit (MCH100110; Merck Millipore). The HN22 and HSC4 cells were seeded on 6-well plates for 24 h and then treated with various concentrations (0, 5, 7.5 and 10 µM) of Ordn for 48 h. The harvested cells were washed with PBS and collected by centrifugation at 1,500 × g for 5 min at room temperature. Following centrifugation, the supernatant was removed and the cell pellets were stained with the Muse™ MitoPotential working solution for 20 min at 37°C. After the cells were stained with 7-aminoactinomycin D (7-AAD) for 5 min at room temperature, the stained cells were examined using the Muse™ cell analyzer.

The assay was carried out using the Muse™ Multi-caspase assay kit (MCH100109; Merck Millipore) to assess the activation of multiple caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9). Briefly, HN22 and HSC4 cells were seeded at 37°C in a 6-well plate for 24 h. After treatment with various doses of Ordn (0, 5, 7.5 and 10 µM), the cells were washed in PBS and resuspended in 1X caspase buffer. Muse™ Multi-Caspase reagent working solution was added to the cells and kept incubated for 30 min at 37°C. One hundred and fifty microliters of Muse™ Caspase 7-AAD working solution was added in each tube and incubated for 5 min at room temperature. The data were analyzed using the Muse™ cell analyzer.

The cells were harvested and washed with cold PBS. Cell lysates was carried out using RIPA lysis buffer and the lysate was then subjected to centrifugation at 16,000 × g for ~30 min at 4°C. Total protein concentrations in the supernatant were determined through calibration with BSA. Total protein extracts were separated electrophoretically using 10, 12 or 15% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes. After the transfer, the membranes were blocked for ~2 h at room temperature with skim milk. The membranes incubated overnight at 4°C with antibodies (all diluted 1:1,000) against CHOP, DR4, DR5, PARP, C-PARP, p38, p-p38, JNK, p-JNK, p21, p27, Mcl-1, survivin, tBid, Bax, cyto c, COX4, α-tubulin, Apaf-1 and actin. After washing 5 times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody [goat anti-rabbit IgG (#31460, 1:6,000 dilution), goat anti-mouse IgG (#31430, 1:5,000 dilution) (both from Thermo Fisher Scientific, Waltham, MA, USA) and donkey anti-goat IgG (sc-2020, 1:4,000 dilution; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Immunoblotting was performed using the ECL Plus Western blotting detection system (Santa Cruz Biotechnology, Inc.) and then each protein was quantified by ImageJ Instrument software.

The data are presented as the means ± SD. Statistical analysis of the data was performed using the Prism 5.0 statistical package. The statistical significance of differences among groups were analyzed using ANOVA and Fisher's Least Significant Difference post hoc test. Mean values were considered statistically significant at P<0.05. In the present study, the data are representative of 3 independent experiments in triplicate.

To examine the effects of Ordn on the viability of the oral cancer cell lines, HN22 and HSC4, we performed MTT assay, measuring the activity of mitochondrial dehydrogenases (28). We treated the cells with Ordn for different periods of time (24 or 48 h) and various concentrations. As a result, Ordn significantly decreased the viability of both the HN22 (Fig. 1B) and HSC4 (Fig. 1C) cells in a dose- and time-dependent manner. As shown in Fig. 1B and C, the IC₅₀ values of Ordn were determined from the dose-response curves of the HN22 and HSC4 cells, accounting for 6.5 and 8.6 µM in the HN22 and HSC4 cells, respectively. Treatment of the HN22 cells with Ordn at 5, 7.5 and 10 µM for 48 h decreased cell viability to 60.97, 35.81 and 25.14% relative to that of the control, respectively. Similarly, the viability of the HSC4 cells treated with Ordn at 5, 7.5 and 10 µM for 48 h dose-dependently decreased to 81.77, 62.02 and 37.81% relative to that of the control, respectively. Morphological changes were examined under an optical microscope following treatment of the oral cancer cells with Ordn at concentrations of 0, 5, 7.5 and 10 µM for 48 h. As shown in Fig. 1D, it was found that the control cells exhibited normal cell shapes with a clear outline and were spread evenly in the culture plates. Following 48 h of treatment with Ordn, a significant proportion of the oral cancer cells became dislodged from the plates. In addition, the remaining adherent oral cancer cells exhibited typical morphological changes, such as cell shrinkage, floating and large intercellular spacing. Ordn markedly suppressed colony formation in both the HN22 and HSC4 cells in a concentration-dependent manner (Fig. 1F and G). Ordn (2 µM) inhibited colony formation by 48 and 43.14% in the HN22 and HSC4 cells, respectively.

To investigate chromatin condensation, fragmented nuclei and nuclear shrinkage, the nuclei of the Ordn-treated cells were observed after DAPI staining. The DAPI-stained nuclei of the HN22 and HSC4 cells were observed using fluorescence microscopy. The DAPI-stained cells were quantified and the apoptotic cell numbers were assessed as means with standard deviation by the graph. The results revealed apoptotic nuclei in the HN22 and HSC4 cells following Ordn treatment at concentrations of 5, 7.5 and 10 µM for 48 h. The percentage of apoptosis was increased to 9.34, 24.44 and 43.18% in the HN22 cells and 10.83, 26.58 and 50.71% in the HSC4 cells with the increasing concentrations of Ordn at 5, 7.5 and 10 µM, respectively (Fig. 1E). To investigate the mechanisms responsible for Ordn-induced apoptosis, the HN22 and HSC4 cells were examined using the Cell Cycle kit and Annexin V and Dead cell kit in Muse™ cell analyzer. The HN22 and HSC4 cells were treated with 0, 5, 7.5 and 10 µM Ordn for 48 h. We found that Ordn resulted in a significant concentration-dependent cell cycle arrest in the sub-G1 phase. The cell cycle distribution in the sub-G1 phase was 1.93±0.47, 12.43±0.21, 31.83±1.74 and 43.13±0.93% in the HN22 cells treated with Ordn at 0, 5, 7.5 and 10 µM, respectively (Fig. 2A). The sub-G1 phase distribution in the HSC4 cells was 3.97±0.15, 28.65±2.62, 29.17±1.88 and 48.10±2.99% in the cells treated with Ordn at 0, 5, 7.5 and 10 µM, respectively (Fig. 2B). To examine cell apoptosis, untreated or Ordn-treated HN22 and HSC4 cells were stained with Annexin V/7-AAD. As shown in Fig. 2C and D, treatment of the cells with Ordn at various concentrations (0, 5, 7.5 and 10 µM) resulted in a dose-dependent increase in the early and late apoptotic population (4.3±0.4, 11.29±0.13, 24.27±0.99 and 58.13±1.88% in the HN22 cells, and 3.65±0.06, 21.61±0.55, 23.24±1.47 and 36.2±2.64% in the HSC4 cells).

As reported previously, the increased generation of ROS can induce cell apoptosis (12,29). Thus, we measured the intracellular ROS levels using the Muse™ cell analyzer with the Muse™ Oxidative Stress kit. As shown in Fig. 3A and B, a marked increase in ROS levels was observed in the cells treated with Ordn at 0, 5, 7.5 and 10 µM for 48 h. In the HN22 cells, we observed a significant increase in ROS production, of 2.71±0.56, 5.98±0.52, 14.04±1.29 and 23.96±3.08% (M2 phase of ROS positively stained cells) at Ordn concentrations of 0, 5, 7.5 and 10 µM, respectively (Fig. 3A). For the HSC4 cells, the obtained results were 5.01±0.36, 9.67±0.96, 16.01±2.67 and 29.02±2.65% of the M2 phase cells, respectively (Fig. 3B). We then examined the protective effects of NAC in the Ordn-treated HN22 and HSC4 cells. NAC is widely used as a free radical scavenger (30). The cells were pretreated with 3 mM NAC, followed by the addition of Ordn at 10 µM for 48 h. As shown in Fig. 3C and D, the loss of cell viability induced by Ordn was prevented by NAC. Moreover, western blot analysis of the HN22 and HSC4 cells revealed that Ordn enhanced the cleavage of PARP; however, pretreatment with NAC reversed these effects (Fig. 3E and F).

The MAPK pathways are one of the numerous cascades downstream of the ROS signaling pathway closely associated with apoptosis, as previously reported (31). In this study, we carried out western blot analysis to determine whether Ordn can induce the activation of the MAPK signaling pathway. Therefore, we examined the changes in the expression of proteins associated with the MAPK pathway, including p38 and JNK in the oral cancer cells following treatment with Ordn. The phosphorylation levels of p38 and JNK were markedly increased in response to Ordn treatment in the HN22 and HSC4 cells (Fig. 4).

A previous study provided evidence that ROS generation is increased in ER stress (32). In addition, a close association has been identified between DR4 and DR5 expression and ER stress (20). As CHOP is an ER stress-inducible transcription factor (20), in this study, we examined whether Ordn treatment induces ER stress in the HN22 and HSC4 cells. Using western blot analysis, we examined whether the CHOP, DR4 and DR5 protein levels were upregulated following treatment of the cells with Ordn. Ordn treatment increased CHOP levels in the oral cancer cells, preceding the upregulation of the DR4 and DR5 levels (Fig. 5). To further characterize the molecular mechanisms responsible for Ordn-induced apoptosis, the expression levels of cell cycle modulators (p21 and p27), pro-apoptotic proteins (tBid and Bax) and anti-apoptotic proteins (Mcl-1 and survivin) were detected in the HN22 and HSC4 cells treated with Ordn. We found that Ordn decreased the expression of Mcl-1 and survivin, and increased p21, p27, tBid and Bax expression (Fig. 6). The Loss of mitochondrial inner trans-membrane potential is a reliable indicator of mitochondrial dysfunction (21). This phenomenon is associated with the early stages of apoptosis (33). In this study, following treatment of the cells with Ordn, the state of mitochondrial membranes was assessed using a Muse™ cell analyzer. Due to the accumulated fluorescent dye within inner membrane of intact mitochondria, control cells emit a high fluorescence intensity (34). Treatment of the cells with Ordn at a high concentration led to a decrease in fluorescence. Following treatment with Ordn at concentrations of 0, 5, 7.5 and 10 µM, the percentage of depolarized HN22 cells was 2.41±1.03, 18.36±2.67, 45.95±1.42 and 59.32±1.02%, respectively (Fig. 7A). The HSC4 cells exhibited a depolarized population of 2.47±0.41, 14.00±0.53, 19.72±2.00 and 40.10±8.75% at concentrations of 0, 5, 7.5 and 10 µM Ordn, respectively (Fig. 7B). Furthermore, we examined the changes in the expression of downstream molecules that can occur after the loss of MMP. Firstly, we analyzed the release of cyto c as an apoptosis-related mitochondrial downstream molecule by western blot analysis. The cyto c protein is the pro-apoptotic mitochondrial protein located in the intermembrane space (35). Our data indicated that the amount of cyto c in the cytoplasm increased as a result of mitochondrial release in the HN22 and HSC4 cells treated with Ordn (Fig. 7C and D). During the apoptotic cascades, Apaf-1 and PARP play an important role (35). Thus, in this study, the expression levels of Apaf-1, PARP and cleaved PARP in the oral cancer cells treated with Ordn were examined by western blot analysis. As shown in Fig. 7C and D, the expression levels of Apaf-1 and cleaved PARP were increased significantly, whereas the expression of PARP was decreased in the HN22 and HSC4 cells treated with Ordn. These results indicated that Ordn regulated these proteins in a concentration-dependent manner. It is well known that the release of cyto c from the mitochondria can trigger a cascade of caspases, which is associated with the final pathway of cell apoptosis (36). The Muse™ Multi-Caspase assay kit was used to detect the presence of multiple caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9) apart from caspase-2. To determine whether caspase plays a role in the Ordn-mediated apoptosis of oral cancer cells, the HN22 and HSC4 cells were examined using the Muse™ cell analyzer after Muse™ Multi-Caspase Reagent and Muse™ Caspase 7-AAD staining. The number represents the percentage of cells with caspase activity (lower right quadrant), and cell population of caspase activity/dead cells (upper right quadrant) in each condition. Multi-caspase activity was activated in the HN22 and HSC4 cells depending on the concentration of Ordn (Fig. 8). The results indicated that the apoptosis of oral cancer cells was induced by Ordn via the activation of caspases.