The following reagents were used: Crocetin (MP Biomedicals, Solon, OH, USA; C₂₀H₂₄O₄; formula weight, 328.4; purity, >98%), docetaxel (Jiangsu Hengrui Medicine Co., Ltd., Nanjing, China), trypsin (Gibco, Carlsbad, CA, USA), fetal bovine serum (Gibco), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; MP Biomedicals, Santa Ana, CA, USA) and TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). The Rh123 and Hoechst 33258 dyes were purchased from Sigma (St. Louis, MO, USA), and an inverted microscope obtained from Olympus (Tokyo, Japan) was used. Cleaved caspase-3 and cytochrome c antibodies were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). Horseradish peroxidase-conjugated anti-rabbit antibody was shipped from Boster (Wuhan, China).

Crocetin was diluted in phosphate-buffered saline (PBS), filter sterilized (GE Healthcare, Greenwich, CT, USA) and stored at 4°C or −20°C (for a maximum of 2 months) in the dark.

The GC BGC-823 cell line was obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The GC cell line was cultured in RPMI-1640 medium supplemented with 10% fetal-bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. The cell culture dish was placed in a humidified atmosphere with 5% CO₂ at 37°C. The medium was changed every 2–3 days and the cells were trypsinized, harvested and seeded in a novel plate once the cells had reached 80–90% confluence.

The effect of crocetin on cell proliferation was determined by the MTT uptake method. The cells (1×10⁵/ml/well) were incubated with varying concentrations of crocetin in a 96-well plate and then incubated for 24, 48 or 72 h at 37°C. MTT solution (10 μl) was added to each well and incubated for 4 h at 37°C. Subsequently, the medium was replaced by 150 μl dimethylsulfoxide (DMSO) per well to dissolve the formazan crystals. The absorbance value of each well was determined by the enzyme-linked immunosorbent assay (wavelength, 570 nm).

The following groups were used: Group A, the blank control group; group B, the 200-μmol crocetin group; group C, the docetaxel 5-μmol group (18); and group D, the 0.5% DMSO group. The four groups were supplemented with 2×10⁶/ml BGC-823 cells for 48 h, and changes in cell morphology were observed by inverted fluorescence microscopy (Leica DMI4000B, Leica, Mannheim, Germany).

DNA staining by Hoechst 33258 dyes were used to evaluate the chromosomal condensation and morphological changes. The BGC-823 cells were plated in 6-well plates. Following treatment, the BGC-823 cells were fixed with 4% paraformaldehyde for 20 min. Hoechst 33258 (5 mg/ml in PBS; pH 7.4) was added to the cells for 10 min, followed by washing with PBS three times. The nuclear morphology was observed using a fluorescence microscope (Leica DMI4000B). The intensity of the mean fluorescence was analyzed using ImageJ 1.41o software (NIH, Bethesda, Maryland, USA).

The MMP was measured using Rh123, a cationic fluorescent dye, as previously described (19). In the four groups, Rh123 was added to the cultures to a final concentration of 2 μM for 30 min at 37°C. Following incubation, the cells were rinsed with PBS and images were captured by Confocal Laser Scanning Microscopy (LSM 710; Zeiss, Oberkochen, Germany) immediately afterwards. The intensity of the mean fluorescence was analyzed using ImageJ 1.41o software (NIH).

To confirm the apoptosis mechanisms induced by crocetin in the BGC-823 cells, the processing of cytochrome c and cleaved caspase-3 was analyzed by western blotting. The cells were treated as aforementioned and then washed three times with cold PBS (pH 7.4) and lysed in ice-cold lysis buffer [1X PBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate and 1% phenylmethanesulfonyl fluoride (PMSF)]. The lysate was centrifuged at 12,000 × g for 5 min at 4°C, then the supernatant was collected and the protein concentration was quantified using a bicinchoninic acid protein assay kit (Bioworld, Dublin, OH, USA). Equal quantities of protein were separated on 15% SDS-polyacrylamide gels (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated overnight with different primary antibodies for cytochrome c (1:1,000) and cleaved caspase-3 (1:1,000) at 4°C. Following three washes with TBST, the blots were incubated with the secondary horseradish peroxidase-conjugated anti-rabbit antibody at room temperature for 1 h followed by washing again three times using TBST buffer. Subsequently, the antibody-bound proteins were detected by an Enhanced chemiluminescence substrate (Vector Labs, Burlingame, CA, USA) and exposed to X-ray films. ImageJ 1.41o software (NIH) was used to quantitatively analyze the expression levels of the protein.

All the experiments were repeated three times. All the data are presented as the mean ± standard error of the mean. Statistical significance was determined using a one-way analysis of variance followed by Duncan’s multiple range test, utilizing a SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). The differences between groups were compared with the least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay demonstrated that crocetin significantly inhibited the viability of the BGC-823 cells (Fig. 1). The cells were incubated in the absence or presence of various concentrations of crocetin (25–300 μM) for 24, 48 and 72 h. The MTT assay revealed that the crocetin-treated cells exhibited a significant reduction in cell proliferation in a concentration- and time-dependent manner, and that the MTT absorbance value was significantly reduced, demonstrating a significant difference compared with the control group (P<0.05). The 50% maximal inhibitory concentration (IC₅₀) was 200 μM in the 48 h group (Fig. 1).

The cells treated with vehicle (Fig. 2A) were healthy, as they had networks of cell processes and vacuole-free cell bodies. Connections between BGC-823 cells were clearly observed. Nucleus fragmentation and shrinkage of the cell bodies were observed when the BGC-823 cells were exposed to 200 μM crocetin (Fig. 2B) or docetaxel (Fig. 2C) for 48 h. DMSO itself did not interfere with the BGC-823 cells (Fig. 2D).

The nuclear morphological changes associated with apoptosis were observed by Hoechst 33258 staining. The control group (Fig. 3A and E) revealed intact and relatively large nuclei, whereas the crocetin- or docetaxel-treated BGC-823 cells exhibited an increase in condensed nuclei (Fig. 3B, C and E). DMSO itself did not interfere with the BGC-823 cells (Fig. 3D and E).

In this study, the effects on the 200 μM crocetin-induced changes of the MMP in the BGC-823 cells were investigated with the fluorescent dye, Rh123, a cell permeable cationic dye, which preferentially enters into the mitochondria based on the highly negative MMP. Following the exposure to 200 μM crocetin for 48 h, the mitochondrial uptake of Rh123 was significantly reduced (Fig. 4B) and the intensity of the mean fluorescence was significantly decreased (P<0.01) by 50% compared with the control (Fig. 4E).

Cytochrome c is a component of the electron transport chain in the mitochondria, and it is also involved in the initiation of apoptosis. Therefore, the levels of cytochrome c and cleaved caspase-3 in cortical neurons were examined in the present study by western blotting. The levels of cytochrome c and cleaved caspase-3 were shown to be markedly increased in the cells that were incubated with 200 μM crocetin for 48 h compared with the control group (Fig. 5A–D).