Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), naphthazarin (Fig. 1) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution and a lactate dehydrogenase (LDH) assay were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis and caspase-3 activation kits were purchased from KeyGen Biotech Co., Ltd., (Nanjing, China).

The human colorectal cancer SW480 cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM (Sigma-Aldrich; Merck Millipore) supplemented with 10% FBS in a humidified atmosphere of 5% CO₂ at 37°C. A total of 1×10⁴ cells/well were seeded in a 96-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1, 5, 10 and 20 µM naphthazarin for 24 h. Subsequently, 20 µl MTT solution (Sigma-Aldrich; Merck Millipore) was added to each well and incubated at 37°C in a 5% CO₂ incubator for 4 h. Following incubation, the culture medium was replaced and 200 µl DMSO was added to each well and agitated for 20 min at room temperature. Cell viability was analyzed at a wavelength of 490 nm using an ELISA reader (Multiskan EX; Thermo Labsystems, Helsinki, Finland). .

SW480 cells (1×10⁴ cells/well) were seeded in a 96-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. After incubation, SW480 cells were treated with to 0, 0.5, 1, 5, 10 and 20 µM naphthazarin for 24 h. Subsequently, 100 µl LDH solution was added to each well and cultured for 30 min. The absorbance was read at a wavelength of 490 nm using an ELISA reader (Multiskan EX; Thermo Labsystems).

SW480 cells (1×10⁶ cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were trypsinized (Sangon Biotech Co., Ltd., Shanghai, China), washed with phosphate-buffered saline (PBS) and fixed in precooling 75% ethanol at 4°C overnight. Next, Annexin-V FITC and PI were added and incubated for 10 min at room temperature in the dark. Flow cytometry analysis was performed on a FACScan flow cytometer (BD Biosciences, San Diego, CA, USA) using emission filters of 525 and 575 nm. Data were analyzed using CellQuest Pro software version 5.1 (BD Biosciences).

SW480 cells (1×10⁶ cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. Following incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were then incubated with 0.1% sodium citrate containing 0.1% Triton X-100 (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at 4°C. Cell nuclei were observed using a fluorescent microscope (Zeiss Axio Observer A1; Carl Zeiss, Inc., Oberkochen, Germany).

SW480 cells (1×10⁶ cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were harvested with PBS and extracted in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the by Pierce BCA protein assay kit (BD Biosciences). Equal amounts of protein were resolved on 6–15% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Membranes were then incubated with antibodies against poly (ADP-ribose) polymerase (PARP; 1:1,000; D161071; Santa Cruz Biotechnology, Inc.), Bax (1:500; AF0057; Beyotime Institute of Biotechnology), Bcl-2 (1:500; AF0060; Beyotime Institute of Biotechnology) and β-actin (1:500; AA128; Beyotime Institute of Biotechnology) overnight at 4°C. Membranes were next washed with TBS containing Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:2,000; Sangon Biotech, Co., Ltd.) at 37°C for 2 h. The protein band was detected using ImageLab 3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

SW480 cells (1×10⁶ cells/well) were seeded in a 6-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. After incubation, SW480 cells were treated with 0, 0.5, 1 and 5 µM naphthazarin for 24 h. SW480 cells were harvested with PBS and extracted in cold RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the Pierce BCA protein assay kit (BD Biosciences). Equal amounts of the total protein were mixed with Ac-DEVD-pNA (Beyotime Institute of Biotechnology) for caspase-3 expression and incubated at 37°C for 2 h in the dark. Caspase-3 activation was analyzed at a wavelength of 405 nm using an ELISA reader (Multiskan EX; Thermo Labsystems).

Data are presented as the mean ± standard deviation of three independent experiments. Differences between groups were analyzed using the Student's t-test and the SPSS 17.0 program (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of naphthazarin on cell viability was investigated by MTT assay in SW480 cells. Following treatment with 5, 10 and 20 µM naphthazarin significantly decreased cell viability of SW480 cells in a dose-dependent manner (P<0.01; Fig. 2). These results indicate that naphthazarin exhibits an anticancer effect on human colorectal cancer cells.

A LDH assay was performed to investigate the effect of naphthazarin on SW480 cell cyotoxicity. Naphthazarin significantly increased cytotoxicity of SW480 cells in dose-dependent manner following treatment with 5, 10 and 20 µM naphthazarin for 24 h (Fig. 3).

The effect of naphthazarin on SW480 cell apoptosis was investigated. Upon treatment with 1 and 5 µM naphthazarin for 24 h, the rate of cell apoptosis was significantly increased in SW480 cells in a dose-dependent manner (Fig. 4).

SW480 cells were stained with DAPI and observed under a fluorescent microscope to investigate the effect of naphthazarinon nuclear apoptosis. Nuclear apoptosis was observed in SW480 cells following treatment with 0.5, 1 and 5 µM naphthazarin for 24 h (Fig. 5).

To determine whether PARP is associated with the effects of naphthazarin on cell viability and apoptosis in SW480 cells, PARP protein expression was evaluated by western blotting. As shown in Fig. 6, treatment with 0.5, 1 and 5 µM naphthazarin for 24 h decreased PARP protein expression, however, no significant differences were observed when compared with the control group.

To further investigate the anticancer effect of naphthazarin, the protein expression of Bcl-2 and Bax in human colorectal SW480 cancer cells was analyzed by western blotting following naphthazarin treatment (Fig. 7A). The results revealed that treatment with 5 µM naphthazarin for 24 h significantly decreased Bcl-2 expression in cells compared with the control (P<0.01; Fig. 7B). Furthermore, treatment with 1 and 5 µM naphthazarin for 24 h significantly increased Bax protein expression in a dose-dependent manner compared with the control (Fig. 7C).

To confirm that potential mechanism of naphthazarin on cell apoptosis of human colorectal cancer cell, we also examined the activation of caspase-3 after naphthazarin treatment with for 24 h. Treatment with 5 µM naphthazarin resulted in significantly increased levels of caspase-3 activation in SW480 cells compared with the control (Fig. 8).