The DB used in the present study was obtained from China-Japan Union Hospital chemical laboratory and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium was purchased from Gibco Life Technologies (Carlsbad, CA, USA) and fetal bovine serum was purchased from HyClone Corp. (Logan, UT, USA). The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA): Rabbit polyclonal cyclin-dependent kinase 2 (Cdk2; sc-163; 1:200), mouse monoclonal transformation related protein 53 (p53; sc-126; 1:200), rabbit polyclonal cyclin-dependent kinase inhibitor 1A (p21; sc-397; 1:200) and rabbit polyclonal checkpoint kinase 1 (Chk1; sc-7898; 1:200). Goat anti-mouse (sc-2005) and anti-rabbit (sc-2054) immunoglobulin G horseradish peroxidase-conjugated secondary antibodies were used (1:1,000; Santa Cruz Biotechnology, Inc.). The anti-β-actin antibody was purchased from Promega Corporation (Madison, WI, USA).

The B16 mouse melanoma cell line was maintained in RPMI-1640 medium, containing 10% fetal bovine serum, in an incubator with 5% CO₂ at 37°C. Trypsin (0.25%) was used to digest the cells.

B16 cells in the logarithmic growth phase were seeded into 96-well plates at ~5×10⁴ cells/ml. Following incubation for 24 h at 37°C, the cells were exposed to 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µg/ml of DB (the experimental groups) or remained untreated (control), with four wells for each group. The cells were further divided into 24 and 48 h treatment groups. Every 24 h, MTT (20 µl of a 5 µg/ml solution) was added to the wells containing the cultured cells, which were then incubated for 4 h. Following incubation at 37°C, the culture medium was removed and 150 µl dimethyl sulfoxide was added. The absorbance (A) was measured using a microplate reader (Bio-Rad 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm, and the rate of the inhibition of cell growth was calculated using the following formula: Cell growth inhibition rate (%) = (1 − Aexperimental group / Acontrol group) × 100.

B16 cells in the logarithmic growth phase were digested with trypsin and seeded into 96-well plates with 100 µl/well suspension containing ~5×10⁴ cells/ml. Following culture for 24 h, the media was discarded. The doses of DB and the incubation durations were as described for the MTT assay above. The morphology of the cells was observed using an inverted phase contrast microscope (BX41-PHD-P11; Olympus, Numazu-shi, Japan).

The mRNA expression was measured in the B16 cells in the logarithmic growth phase following treatment with DB (0.5, 1.0 and 2.0 µg/ml) for 24 or 48 h. The cells were digested with 0.25% trypsin and pelleted by centrifugation at 2,000 × g for 5 min at 37°C. The total RNA was extracted with TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) and the concentration and purity of the RNA samples were determined by measuring the A260 and A280 optical density values using an ultraviolet (UV) spectrophotometer (UV-2401PC; Bayer AG, Leverkusen, Germany). The cDNA (100 ng) was obtained by RT of 1 µg mRNA with SMART^® MMLV Reverse Transcriptase (639523; Takara Bio, Inc., Otsu, Japan). The PCR kit used was 2XEasyTaq PCR SuperMix (AS111-02; Beijing TransGen Biotech Co., Ltd., Beijing, China). The primer sequences, which were obtained from Sangon Biotech, Co., Ltd. (Shanghai, China), were as follows: p53, upstream 5′-CATGGACGATCTGTTGCTG-3′ and downstream 5′-TCGGGTGGCTCATAAGGT-3′; p21, upstream 5′-AGCTCAATGGACTGGAAGGG-3′ and downstream 5′-GAGCTGGAAGGTGTTTGGGG-3′; Cdk2, upstream 5′-GTTGACGGGAGAAGTTGTGG-3′ and downstream 5′-GAAGGACACGGTGAGAATGG-3′; Chk1, upstream 5′-CTTTGGGAGAAGGTGCCTAT-3′ and downstream 5′-ATGCCGAAATACCGTTGC-3′. β-actin was used as an internal control to assess the quality of the cDNA templates. The sequences of the β-actin primers were as follows: Upstream 5′-GTAAAGACCTCTATGCCAACACA-3′ and downstream 5′-GGACTCATCGTACTCCTGCTTG-3′. The sizes of the amplified fragments for p53, p21, Cdk2, Chk1 and β-actin were 543, 485, 302, 431 and 226 bp, respectively. The conditions used for qPCR of the p53, p21, Cdk2, and Chk1 genes were as follows: 95°C for 5 min; 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec and 72°C for 5 min. The qPCR products were visualized using a camera and a digital gel imaging system (GIS; Tanon 2500R; Tanon Science & Technology Co., Ltd., Shanghai, China). GIS was used to determine the grayscale values of the electrophoretic bands for p53, p21, Cdk2 and Chk1. The expression levels of these genes were expressed as a relative ratio of mRNA against the mRNA of β-actin.

Western blotting was used to determine the protein expression levels in the B16 cells in the logarithmic growth phase following exposure to DB (0.5, 1.0 or 2.0 µg/ml) for 24 or 48 h. Following treatment with DB, the cells were washed with phosphate-buffered saline (PBS) and centrifuged at 2,000 × g for 5 min. Following centrifugation, protein homogenization buffer (100 µl), β-mercaptoethanol (1.2 µl) and 200 mmol/l phenylmethylsulfonyl fluoride (0.3 µl) were added to the resuspended cells. The cells were ruptured using an ultrasonic homogenizer, and the protein concentrations were determined using a microplate reader. Protein (40 µg) from each sample was electrophoresed and the proteins were transferred from the 12% SDS-PAGE gel (Beijing Solarbio Science & Technology Co., Ltd., Shanghai, China) onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk prior to being incubated at 4°C overnight with antibodies directed against p53, p21, Cdk2 and Chk1. Following incubation with secondary antibodies for 1 h at room temperature, the membranes were processed and analyzed. The expression levels of p53, p21, Cdk2 and Chk1 were quantified using a color image analysis system (Tanon 4100; Tanon Science & Technology Co., Ltd.).

The B16 cells were incubated with DB for 24 or 48 h, digested with EDTA (Sangon Biotech, Co., Ltd.), harvested and incubated in 70% ethanol at 4°C for 24 h. A total of ~1×10⁶ cells were resuspended in 100 µl PBS. Subsequently, 5 µl propidium iodide (PI) was added and the cells were incubated in PI for 30 min at room temperature in the dark. The cell cycle status was then assessed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).

The data are expressed as the mean ± standard deviation. One-way analysis of variance was used to determine whether differences were present between the groups. All analyses were performed using SPSS 11.5 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effects of different concentrations of DB on the B16 cells was determined by incubating the cells with DB for 24 or 48 h. The results of this analysis are presented in Tables I and II, and Fig. 1. Following 24 or 48 h treatment with DB, proliferation was significantly more inhibited in the cells treated with 0.5 µg/m, compared with the cells of the control groups (24 h, P<0.05; 48 h, P<0.01). Following 24 h exposure to DB, the proliferation of the cells treated with 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µg/ml were inhibited by 11.06, 24.95, 39.23, 63.28, 68.90, 78.54 and 79.87%, respectively (Table I). Following 48 h exposure to DB, the proliferation of cells treated with 0.5, 1.0, 2.0, 4.0 and 8.0 µg/ml groups were inhibited by 38.53, 51.87, 68.32, 80.84 and 86.11%, respectively (Table II). These findings indicated that inhibition of the proliferation of B16 cells by DB is time- and dose-dependent (Tables I and II).

The B16 cells were incubated with different doses of DB, following which their morphology was assessed using an inverted phase contrast microscope. The results revealed that the adherent control cells grew well and were transparent, with good refractive indices and smooth, clear boundaries. In addition, proliferation of the control cells was robust. The effects of treatment with DB on cell morphology manifested slowly. A small number of cells became round and smaller in size following treatment with 1 µg/ml DB for 24 h, and a number of dead, floating cells were observed. Pronounced changes in cell morphology were increasingly apparent with increased doses of DB. Following treatment with 2 µg/ml DB for 48 h, a marked increase in cell death was observed. The viable cells lost their shape and their surfaces were no longer smooth. Visible cell debris was also present (Fig. 1).

The results of the analysis of the cell cycle using flow cytometry are shown in Fig. 2. The area in front of the red peak represents the G0-G1 phases, the area following the red square represents the G2-M phases and the blue diagonal peaks represent the S phase. No cell cycle arrest was observed in the cells in the blank control group during any phase of the cell cycle. However, the cells exposed to DB exhibited arrest, predominantly in the G1 phase. The number of B16 cells arrested in the G1 phase following exposure to 0.5, 1.0 or 2.0 µg/ml DB for 24 or 48 h was dose-dependent (Fig. 2 and Table III).

The effects of treatment with 0.5, 1.0 or 2.0 µg/ml DB on gene expression in the B16 cells was analyzed subsequent to 24 and 48 h exposure. Following treatment for 24 h, the mRNA expression levels of p53, p21 and Chk1 were significantly increased in the cells treated with 1.0 or 2.0 µg/ml DB, compared with the control cells (P<0.05). Following 48 h exposure, the mRNA expression levels of p53 were significantly increased only in cells treated with 2.0 µg/ml DB, while the mRNA expression levels of p21 and Chk1 were significantly increased in cells treated with all three doses of DB (P<0.05; Fig. 3). By contrast, the mRNA expression levels of Cdk2 were significantly decreased in the cells treated with 2.0 µg/ml DB for 24 h and in the cells treated with 1.0 µg/ml or 2.0 µg/ml DB for 48 h (P<0.05; Fig. 3).

The effects of treatment with 0.5, 1.0 or 2.0 µg/ml DB on the B16 cells were analyzed following 24 or 48 h exposure. The protein expression levels of p53, p21 and Chk1 were significantly increased in the cells treated with DB, compared with the control cells (All P<0.05), with the exception of Chk1 in the cells treated with 0.5 µg/ml DB for 24 h. The protein expression of Cdk2 in the cells treated with 2.0 µg/ml DB for 24 or 48 h was decreased significantly, compared with that observed in the control cells (Fig. 4).