The CAL-27 human oral cancer cell line was maintained in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 units/ml; Gibco; Thermo Fisher Scientific, Inc.), streptomycin (100 µg/ml; Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C in 5% CO₂ in a humid atmosphere.

Cell viability rate was determined by MTT assay. EF24 (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) and cisplatin (Sigma Aldrich; Merck KGaA) were dissolved in dimethyl sulfoxide (DMSO) and distilled water, respectively. The final concentration of DMSO was <0.1%. CAL27 cells (density, 2×10⁴) were seeded into each well of 96-well plates and incubated for 24 h, after which the cells were administered with selected doses of EF24 (0.1–30 µM) or cisplatin as a positive control (0.1–30 µM), and the dose-dependent effects of the drugs on the cells were determined. Following a 24-h incubation, 10 ml MTT solution (5 mg/ml) was added to the medium and maintained at 37°C for 4 h. DMSO was used for dissolving formazan crystals, and the absorbance values were measured at a wavelength of 570 nm using a Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). All experiments were performed in quintuplicate for each dose.

DNA fragmentation was detected as a marker of apoptosis using a Cell Death Detection ELISA^Plus kit (Sigma-Aldrich; Merck KGaA), following the manufacturer's protocol. Briefly, 24 h prior to drug treatment, 2×10⁴ CAL27 cells were seeded into each well of 96-well plates. Various concentrations of EF24 or cisplatin were added to the wells followed by a 24-h incubation. Vehicle control cells were treated with an equal amount of DMSO. Absorbance was measured at a wavelength of 405 nm with a Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek Instruments, Inc.).

CAL-27 cells were treated with EF24 or DMSO only for 24 h. For the MAPK/ERK activation assay, cells treated with 1 µM EF24 for 24 h, following which EF24 was removed and 50 ng/ml 12-phorbol-13-myristate acetate (PMA; Sigma Aldrich; Merck KGaA) was used for another 24 h of incubation. At the end of treatment, whole cells lysates were acquired using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Inc.) and centrifuged at 13,000 × g for 10 min at 4°C. Protein concentration of the supernatant was determined using a Pierce Bicinchoninic Acid assay kit (Thermo Fisher Scientific, Inc.). Total proteins (40 µg per lane) were subjected to 12% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes. After incubating with a blocking solution containing 3% bovine serum albumin (Sigma Aldrich; Merck KGaA) and 0.1% Tween-20 in PBS at room temperature for 30 min, the membranes were incubated with the desired primary antibodies at room temperature for 2 h (Table I), followed by incubation with a goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (cat. no. 31460; 1:20,000; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The immunoactive bands were detected using Pierce™ Western Blot Signal Enhancer (Thermo Fisher Scientific, Inc.), and digital chemiluminescence images were analyzed by GeneTools (version 4.1; Syngene, Frederick, MD, USA). The entire western blot analyses were repeated at least three times.

Apoptosis of CAL-27 cells was detected using the in-situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. Briefly, 1×10⁶ CAL-27 cells were treated with 1 µM EF24 for 24 h, or subsequently treated with 1 µM EF24 for 24 h then 50 ng/ml PMA for another 24 h. Following drug administration, cells were fixed with 4% paraformaldehyde (pH 7.4) and incubated with PBS consisting of 0.1% Triton X-100 and 0.1% sodium citrate at 4°C for 5 min. Following this, 50 µl TUNEL reaction mixture was added to the solution, and the cells were incubated at 37°C for 1 h. Following three washes with PBS, cells were stained with DAPI for 5 min at room temperature. Apoptotic cells were identified by blue excitation light at wavelength of 488 nm. Images were captured using a Nikon Eclipse Ti microscope (Nikon Corporation, Tokyo, Japan) at a magnification of ×40.

All experiments were performed in at least triplicate. The results are presented as the mean ± standard deviation. The statistical significance was analyzed by SPSS software (version 20.0; IBM Corp., Armonk, NY, USA) using Student's t-test for the two group comparisons and one-way analysis of the variance for the multi-group comparisons. For multiple comparisons, the Student-Newman-Keuls method was used as a post-hoc test. P<0.05 was considered indicate a statistically significant difference.

Previous studies have demonstrated the cytotoxicity of EF24 in prostate, ovarian and breast cancer cells (11–13). To determine the ability of EF24 to induce apoptosis in oral cancer, human CAL-27 cells were treated with various concentrations of EF24 for 24 h, and cisplatin was used as positive control. As presented in Fig. 1A, treatment with cisplatin at doses from 0.1 to 1 µM did not significantly affect the viability of CAL-27 cells, but cisplatin at 3, 10 and 30 µM significantly reduced CAL-27 cell viability (P<0.05). When the cells were treated with EF24, the growth of CAL-27 cells was inhibited (P<0.05) in a dose-dependent manner from 1 to 30 µM, and the viability under each condition was lower that of cisplatin. Notably, the inhibitory effects were observed at 1 µM EF24, a dose at which cisplatin had no significant effects on cell viability, indicating the higher potency of EF24. Similarly, compared with cisplatin treatment, administration of EF24 elevated the DNA fragmentation rate of CAL-27 cells in a in a dose-dependent manner from 1 to 30 µM (P<0.05), suggesting that EF24 is more effective at inducing the apoptosis of CAL-27 cells than cisplatin (Fig. 1B).

CAL-27 cells incubated with EF24 (1 or 10 µM) for 24 h were assessed by western blot analysis, using antibodies that recognize apoptosis-associated proteins. Protein expression levels of cytochrome c, cleaved caspase 3 and cleaved caspase 9 were significantly increased, compared with non-EF24 treated controls (P<0.05; Fig. 2). Furthermore, the relative expression of cytochrome c, cleaved caspase 3 and cleaved caspase 9 was higher in 10 µM EF24-treated cells, when compared with 1 µM EF24-treated cells. This dose-dependent result indicated that apoptosis induced by EF24 was at least partly via the activation of caspases.

Several studies have demonstrated the important role of the MAPK/ERK signaling pathway in cell cycle arrest and apoptosis in response to stress (14–16). To determine whether the MAPK/ERK signaling pathway is involved in EF24 stimulated apoptosis, the activated and inactivated forms of mitogen activated protein kinase kinase 1 (MEK1) and ERK protein were examined in CAL-27 cells treated with EF24 (1 or 10 µM) for 24 h. Representative results from each condition are presented in Fig. 3. Densitometric analysis indicated that EF24 induces deactivation of the MAPKs in a dose-dependent manner, as revealed by their downregulated phosphorylation (P<0.05). These results suggested that EF24 may inhibit the MAPK/ERK signaling pathway during initiation of oral cancer cell apoptosis.

PMA is a well-known tumorigen that activates protein kinase C isozymes, which are upstream regulators of MAPK signaling pathway (17). The phosphorylation level of MEK1 and ERK in CAL-27 cells in response to treatment with EF24 and PMA were studied. Western blot analysis revealed that sequential administration of EF24 and PMA significantly elevated active MEK1 and ERK levels, when compared with EF24 alone (P<0.05; Fig. 4), indicating that PMA treatment abolished the EF24-induced MAPK/ERK signaling pathway deactivation.

To examine whether the MAPK/ERK activator PMA may rescue EF24-induced apoptosis, CAL-27 cells were treated with DMSO, EF24 alone or a sequential combination of EF24 and PMA. Western blotting results demonstrated that the ratios of cleaved caspase 3 and 9 to GAPDH were significantly decreased with the administration of PMA (P<0.05; Fig. 5A). Similarly, TUNEL staining indicated that cells sequentially treated with EF24 and PMA had a lower percentage of apoptotic (TUNEL-positive) cells, when compared with EF24 alone (P<0.05; Fig. 5B). These results indicated that PMA treatment effectively ameliorated the cytotoxicity of EF24, thus confirming that deactivation of the MAPK/ERK signaling pathway may represent a major underlying cellular mechanism of EF24-induced apoptosis of CAL-27.