The HN22 and HSC4 human OSCC lines were obtained from Dankook University (Cheonan, Korea) and Hokkaido University (Hokkaido, Japan), respectively, and were cultured in Hyclone Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum, and 100 U/ml each of penicillin and streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO₂ in humidified air.

Cell viability was measured using the CellTiter 96™ AQueous assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. HN22 and HSC4 cells were seeded in a 96-well plate for 24 h and then treated with 7,8-DHF (5, 10, 20 and 40 μM) for 24 and 48 h. Cell viability was measured by adding the 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) dehydrogenase enzyme substrate and the electron coupling reagent phenazine methosulfate. The plates were incubated at 37°C in 5% CO₂ for 2 h after a 24 h or 48 h post-treatment with 7,8-DHF. Absorbance at 490 nm was recorded using a GloMax-Multi Microplate Multimode reader (Promega).

The levels of nuclear condensation and fragmentation were observed by nucleic acid staining with DAPI. HN22 and HSC4 cells treated with 7,8-DHF were harvested by trypsinization, and fixed in 100% methanol at room temperature for 20 min. The cells were seeded on slides, stained with DAPI (2 μg/ml), and monitored by FluoView confocal laser microscopy (Fluoview FV10i; Olympus Corp., Tokyo, Japan).

After 48 h of 7,8-DHF treatment, the HN22 and HSC4 cells were collected by centrifugation and combined with adherent cells. The cells were washed with cold phosphate-buffered saline (PBS), pooled, and centrifuged before being fixed in 70% ice-cold ethanol overnight at −20°C, and then treated with 150 μg/ml RNase A and 20 μg/ml PI (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were analyzed, and the distribution of the cells in different phases of the cell cycle was calculated using flow cytometry with a MACSQuant Analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

The cells were seeded on a 100-mm dish containing 5.2×10⁵ cells/well for HN22 cells and 8.8×10⁵ cells/well for HSC4 cells and treated with various concentrations of 7,8-DHF (10, 20 and 40 μM) for 48 h. Both adherent and floating cells were harvested and washed once with PBS. The cells were incubated with Annexin V/7-AAD for 20 min at room temperature in the dark to detect apoptosis, followed by a 6 h incubation at 37°C. Apoptotic and necrotic cells were analyzed by flow cytometry (Muse Cell Analyzer; Merck Millipore, Billerica, MA, USA) using the Muse Annexin V/7-AAD & Dead Cell kit (MCH100105; Merck Millipore). The experiment was performed in triplicate.

Total RNA was extracted from the cells using the TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA), and 2.5 μg of RNA was used to synthesize cDNA using the HelixCript™ first-strand cDNA synthesis kit (NanoHelix, Seoul, Korea). cDNA was obtained by PCR using β-actin-specific and Sp1-specific primers as described below under the following PCR conditions (35 cycles: 1 min at 95°C, 1 min at 56°C and 1 min at 72°C). The β-actin primers were: forward, 5′-GTG GGG CGC CCC AGG CAC CA-3′ and reverse, 5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′; and the Sp1 primers were: forward, 5′-ATG CCT AAT ATT CAG TAT CAA GTA-3′ and reverse, 5′-CCC TGA GGT GAC AGG CTG TGA-3′. PCR products were analyzed by 1% agarose gel electrophoresis.

HN22 and HSC4 cells were seeded onto sterilized glass coverslips on 6-well tissue culture plates for 24 h and incubated with 7,8-DHF for 48 h. The cells were fixed and permeabilized with Cytofix/Cytoperm solution for 30 min. The cells were blocked with 1% bovine serum albumin and then incubated with monoclonal Sp1 and cleaved caspase-3 antibody at 4°C overnight to express Sp1 and cleaved caspase-3. After washing with PBS containing 0.05% Tween-20 (PBST), Sp1 and cleaved caspase-3 antibodies were reacted with a Jackson 488-conjugated anti-mouse and Jackson 647-conjugated anti-rabbit secondary antibody at room temperature for 1 h and mounted with Vectashield mounting medium to assess DAPI fluorescence (Vector Laboratories, Inc., Burlingame, CA, USA). The cells were visualized using a FluoView confocal laser microscope.

This method was previously described (27). Briefly, HN22 and HSC4 cell lysate (500 μg) was reacted with Sepharose 4B or 7,8-DHF-Sepharose 4B (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) matrix beads (0.2 g) in reaction buffer [50 mM Tris, pH 7.5, 5 mM ethylenediamine-tetraacetic acid (EDTA), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.01% Nonidet P-40, 2 μ/ml bovine serum albumin, 0.02 mM phenylmethylsulfonyl fluoride (PMSF) and 1X proteinase inhibitor cocktail]. After overnight incubation with gentle rocking at 4°C, the beads were washed five times with washing buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40 and 0.02 mM PMSF) and proteins bound to the beads were analyzed by western blot analysis.

HN22 and HSC4 cells were treated with 7,8-DHF (10, 20 and 40 μM) for 48 h, washed with PBS, and harvested in ice-cold PRO-PREP™ protein extraction solution (Intron Biotechnology, Inc., Daejeon, Korea) containing a protease inhibitor. The extracted proteins were measured using a Pierce^® BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were separated via 10% or 15% (v/v) SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. After blocking for 2 h at room temperature with 5% non-fat dried milk in PBST containing 0.1% Tween-20, the membrane was incubated overnight at 4°C with the specific antibodies. Enhanced chemiluminescence western blotting was performed according to the manufacturer’s instructions (Thermo Fisher Scientific).

Statistical significance was assessed using the Student’s t-test. P<0.05 relative to the control was considered significant.

The aim of the present study was to investigate the efficiency of 7,8-DHF to inhibit the growth of OSCC cells. The chemical structure of 7,8-DHF is shown in Fig. 1A. To confirm the growth inhibitory effect of 7,8-DHF on OSCC, HN22 and HSC4 cells were treated with 7,8-DHF, and cell viability was determined by the MTS assay. As shown in Fig. 1B, the MTS assay was performed after treatment with 7,8-DHF at various concentrations (5, 10, 20, 30 and 40 μM) for 24 and 48 h. Fig. 1B shows that 7,8-DHF inhibited the viability of OSCC cells in a dose-dependent manner. The changes in the appearance of OSCC cells were observed with an optical microscope after 48 h (Fig. 1C). The apoptotic phenotype was a rounded cell, with cytoplasmic bleeding and an abnormal shape. These results indicate that 7,8-DHF inhibited the growth of human OSCC.

Cancer cell proliferation can be suppressed by apoptosis, inducing cell cycle arrest or both. DAPI staining was performed to confirm the induction of apoptosis by 7,8-DHF in HN22 and HSC4 cells, as DAPI specifically stains nuclei. The results showed fragmented and condensed nuclei in cells treated with 7,8-DHF (10, 20 and 40 μM) for 48 h compared to that in the control (Fig. 2A). The cell cycle distribution was analyzed by FACS analysis. As shown in Fig. 2C, a significant increase in the number of sub-G₁ phase HN22 cells was observed (11.2±1.4% in the presence of 10 μM 7,8-DHF, 31.0±2.3% in the presence of 20 μM 7,8-DHF, and 44.0±2.6% in the presence of 40 μM 7,8-DHF) compared with that of untreated control cells. An increase in the number of sub-G₁ phase HSC4 cells was also observed (5.3±0.3% in the presence of 10 μM 7,8-DHF, 11.5±1.2% in the presence of 20 μM 7,8-DHF and 58.8±1.2% in the presence of 40 μM 7,8-DHF) compared with that in the untreated control cells. Cells stained with Annexin V only were defined as early apoptotic and Annexin V (lower right) and 7-AAD double-stained cells were defined as late apoptotic (upper right). As shown in Fig. 2D, 7,8-DHF displayed marked effects to induce apoptosis of HN22 and HSC4 cells in a dose-dependent manner. Treatment of the HN22 cells with 10, 20 and 40 μM of 7,8-DHF for 48 h resulted in 6.2±1.5, 26.3±2.5 and 19.3±0.9% of early apoptotic cells and 6.1±0.8, 7.1±0.2 and 36.3±0.4% of late apoptotic cells, respectively. Similarly, treatment of HSC4 cells with 7,8-DHF also led to 18.5±2.3, 33.7±0.3 and 22.9±2.2% of early apoptotic cells and 4.2±0.8, 6.9±0.2 and 26.4±1.0% of late apoptotic cells at the same three concentrations as above, respectively. Apparently, 7,8-DHF-mediated apoptosis of HN22 and HSC4 cells, at least in part, contributed to its antiproliferative effects.

As the Sp1 protein plays an important role in oncogenesis, a therapeutic agent that can effectively modulate the Sp1 protein may be a suitable anticancer drug to suppress tumor progression (28). Both HN22 and HSC4 cells were treated with various concentrations of 7,8-DHF (10, 20 and 40 μM) for 48 h to observe Sp1 protein expression levels. Fig. 3A and B shows a significant decrease in Sp1 protein expression levels in the HN22 and HSC4 cells in a dose-dependent manner. We also observed downregulation of the Sp1 protein in HN22 and HSC4 cells at 40 μM 7,8-DHF after different periods of time (0, 12, 24, 36 and 48 h). Additionally, Sp1 mRNA was suppressed by 7,8-DHF in both HN22 and HSC4 cells (Fig. 3C). Consistent with these observations, the immunocytochemistry results revealed decreased Sp1 and increased cleaved caspase-3 levels in a dose-dependent manner in both the HN22 and HSC4 cell lines (Fig. 3D). We conclude that suppression of Sp1 by 7,8-DHF treatment leads to apoptotic cell death. Next, a pull down assay was performed to determine whether 7,8-DHF directly binds to Sp1. Fig. 4A and B showed that 7,8-DHF strongly suppressed Sp1 activity by directly binding to Sp1.

In many previous studies, the downregulation of Sp1 was found to induce apoptosis, and Sp1 was confirmed to be associated with cell apoptosis (28–30). We used western blot analysis to clarify the association between 7,8-DHF and Sp1-mediated apoptosis. We investigated the cell-cycle arrest proteins such as p27 and p21 and found increased levels following 7,8-DHF treatment. We also investigated cell proliferation and survival-related proteins such as cyclin D1, Mcl-1 and survivin, which were decreased in a dose-dependent manner following 7,8-DHF treatment (Fig. 5). We found a decrease in BID and Bcl-_xl and an increase in Bax expression. These proteins were associated with the apoptotic cell death induced by 7,8-DHF. Finally, cleaved caspase-3 and cleaved PARP were induced by 7,8-DHF in a dose-dependent manner (Fig. 6). These results revealed that 7,8-DHF treatment of OSCC decreases Sp1, resulting in growth arrest and apoptotic cell death.