A431 and HaCaT cells were purchased from the Shanghai Cell Collection (Shanghai, China) and the Fuxiang Institute of Biotechnology (Shanghai, China), respectively. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin, in a humidified incubator at 37°C supplied with 5% CO₂. Once cells reached 80% confluence, they were passaged at a 1:3 ratio by dissociation with 0.25% trypsin and 0.02% EDTA. Cells in the logarithmic growth phase were used in all experiments. DMC (>99% purity) was obtained from the YuanYe Institute of Biotechnology (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) to generate a 100 mmol/l stock solution. The stock solution was stored at −20°C and freshly diluted in complete culture medium prior to use. The final concentration of DMSO applied to the cells was <0.1%. Rabbit polyclonal primary antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. AB40415), Bcl-2-associated X protein (BAX; cat. no. AB40636), caspase-3 (cat. no. AB42437), caspase-9 (cat. no. AB32539) and cytochrome c (cat. no. AB133504) were purchased from Biogot Technology Co., Ltd. (Nanjing, China). GAPDH (cat. no. sc-32233) was used as a loading control (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The goat-anti-rabbit secondary antibody (cat. no. sc-362262) was purchased from Santa Cruz Biotechnology, Inc.

The effects of DMC on A431 and HaCaT cell viability were determined using a Cell Counting kit-8 (CCK-8) assay (Vicmed Co., Ltd., Xuzhou, China). Cells were seeded in 96-well plates at a density of 3×10³ cells/well and incubated overnight in DMEM containing 10% FBS. Cells were treated with various concentrations of DMC (0, 5, 10, 20, 40 and 80 µМ) for 24, 48, 72 and 96 h, and were then incubated with CCK-8 reagent for 2 h at room temperature. Absorbance at 450 nm was measured using a microplate spectrophotometer (Thermo Labsystems, Santa Rosa, CA, USA). Inhibition of viability was determined relative to the control group measurement (untreated cells). The IC₅₀ value was calculated using Graphpad Prism software (Graphpad Software, Inc., San Diego, CA, USA). All experiments were performed in triplicate.

Cells were seeded at 1×10⁶ cells/well in 6-well plates, and allowed to grow for 24 h. Cells were treated in triplicate with 0, 5, 10, 20, 40 and 80 µМ DMC for 48 h. Following dissociation with 0.25% trypsin, cells were centrifuged at 2,000 rpm for 5 min at room temperature. The supernatant was discarded, and pellets were washed once with PBS. Cell pellets were resuspended in 500 µl 70% cold ethanol and fixed overnight at 4°C. Following fixation, pellets were washed with PBS, 100 µl RNAse A solution was added, and samples were incubated in a 37°C water bath for 30 min. Subsequently, 400 µl propidium iodide (PI) (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was added, and samples were incubated at 4°C in the dark for 30 min. Samples were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FCS-express version 3 software (De Novo software, Glendale, CA, USA). The experiment was performed in three independent repeats.

A431 and HaCaT cells were cultured in 6-well plates for 12 h and then treated with 0, 5, 10, 20, 40 and 80 µM DMC for 48 h. Cells were dissociated, collected by centrifugation at 200.67 × g for 5 min at room temperature, and washed twice with PBS. Cells (~1×10⁶) were then resuspended in 500 µl binding buffer containing 5 µl Annexin V and 5 µl PI reagents (Nanjing KeyGen Biotech Co., Ltd.). Following a 15 min incubation at room temperature in the dark, the samples were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using the FCS-express V3 software (de novo software, Thornhill, Ontario, Canada).

A431 and HaCaT cells were seeded in 6-well plates and treated with 10, 20, 40 and 80 µM DMC for 48 h. Untreated cells served as control. Following treatment, the medium was discarded, the cells were washed twice with PBS, incubated with Hoechst 33258 (Nanjing KeyGen Biotech Co., Ltd.) for 5–10 min at room temperature in the dark, washed twice with PBS, and finally observed under a fluorescence microscope.

Adherent and floating cells were harvested at 200.67 × g for 5 min at room temperature and washed twice with ice-cold PBS. Cells were homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 100 mM PMSF. Homogenates were centrifuged at 15,000 × g at 4°C for 20 min and the supernatants were collected. Protein concentrations were measured using the bicinchoninic acid method (Nanjing KeyGen Biotech Co., Ltd.), using bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to generate the standard curve. A total of 100 µg protein of each sample was loaded in SDS sample buffer and heat denatured at 100°C for 5 min. Protein samples were then electrophoretically separated by 10–15% SDS-PAGE and transferred onto nitrocellulose membranes (Promega Corporation, Madison, WI, USA). Blocking was performed for 1 h at room temperature in blocking buffer containing 5% non-fat dry milk. Membranes were then incubated with primary antibodies at 4°C overnight (1:1,000 dilution), followed by horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h (1:5,000 dilution). Signals were detected with SuperSignal enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.). All experiments were performed in triplicate. Densitometric analysis was performed on protein bands using ImageQuant^™ TL software version 8.1; Molecular Dynamics; GE Healthcare Life Sciences, Chalfont, UK) (18).

Data were expressed as the mean ± standard deviation. Statistical significance was determined by independent samples t-test and one-way analysis of variance followed by the Tukey's post-hoc test, as appropriate, using SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Statistical graphs were drawn using GraphPad prism (Graph Pad Software, Inc.).

In order to investigate the effect of DMC treatment on A431 and HaCaT cell viability, A431 and HaCaT cells were exposed to various concentrations of DMC for 24, 48, 72 or 96 h, and then analyzed using a CCK-8 assay. As demonstrated in Fig. 2, the cell viability inhibitory rate in DMC-treated cells was significantly increased compared with control cells (P<0.05). The half maximal inhibitory concentration values of DMC for A431 and HaCaT cells at 48 h were 9.2 and 16.22 µM, respectively (Fig. 3). These results indicated that DMC treatment decreased viability of A431 and HaCaT cells in a dose-dependent manner.

To further analyze the mechanism by which DMC inhibits cell viability, A431 and HaCaT cells were treated with 5, 10, 20, 40 and 80 µM DMC for 48 h and then analyzed for cell cycle phase distribution by flow cytometry. The results demonstrated that the percentage of cells in the G₀/G₁ phase was markedly decreased, whereas the percentage of cells in the G2/M phase was markedly increased in the DMC-treated cells compared with untreated cells (Fig. 4). These results indicated that cell cycle arrest at G₂/M may contribute to the inhibitory effects of DMC on cell viability.

A431 and HaCaT cells were treated with 5, 10, 20, 40 and 80 µM DMC for 48 h and apoptosis was measured by Annexin V/PI double staining and flow cytometric analysis. The apoptotic rate of DMC-treated cells was significantly increased in a dose-dependent manner compared with untreated cells (P<0.05; Fig. 5).

To confirm apoptosis by cell morphological observation, A431 and HaCaT cells were exposed to 0, 10, 20, 40 and 80 µM DMC for 48 h, stained with Hoechst 33258, and observed by fluorescence microscopy. Control untreated cells displayed nuclei with uniform staining (Fig. 6). Conversely, shrinkage of the nuclei was observed in the cells exposed to 20–80 µM DMC for 48 h (Fig. 6). The number of visibly apoptotic nuclei increased with increasing concentrations of DMC, and various morphological features of apoptosis were observed, including reduction of cell numbers, nuclei with intensely bright staining and fragmented nuclei (Fig. 6).

In order to explore the mechanisms by which DMC treatment may induce apoptosis, the effects of DMC on the protein expression levels of various established apoptotic markers were examined. A431 and HaCaT cells were treated with 10, 20, 40 and 80 µM DMC for 48 h and then analyzed by western blotting for protein expression of Bcl-2, BAX, caspase-3, caspase-9 and cytochrome c expression (Figs. 7 and 8). As demonstrated in Fig. 7, DMC treatment resulted in a decrease in the protein expression levels of Bcl-2, and an increase in BAX expression (Fig. 7). In addition, DMC treatment caused an increase in the protein expression levels of caspase-9, caspase-3 and cytochrome c in a dose-dependent manner (Fig. 8).