Podophyllotoxin, MTT, propidium iodide (PI), Hoechst 33258 and Rodamine 123 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxycamptothecin (HCPT) was purchased from Harbin Shengtai Pharmaceutical Co., Ltd. (Harbin, China); RPMI 1640 culture medium was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China) and trypsin was purchased from Gibco-BRL (Rockville, MD, USA). Additionally, mouse anti-human β-actin, cytochrome c (cyt-c), caspase-9 and caspase-3 polyclonal antibodies, alkaline phosphatase (AP)-conjugated goat anti mouse polyclonal antibody, SDS-PAGE sample loading buffer, blocking buffer, TBST, buffer5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT) alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, Haimen, China); detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA).

The CKX 41 inverted fluorescence microscope was purchased from Olympus (Tokyo, Japan) and the mini electrophoresis meter and microplate reader were purchased from Bio-Rad (Hercules, CA, USA). The EPICS XL flow cytometer was purchased from Beckman Coulter (Brea, CA, USA); the SP2 laser confocal scanning microscope was purchased from Leica (Solms, Germany) and the CO-150 CO₂ incubator was purchased from New Brunswick Scientific (Edison, NJ, USA).

The human GC SGC-7901 cell line was provided by the Center of Research and Development on Life Sciences and Environmental Sciences of Harbin University of Commerce (Harbin, China). SGC-7901 cells were grown in RPMI 1640 culture medium containing 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO₂.

Exponentially growing cells were washed, digested with trypsin and suspended in RPMI 1640 medium until a concentration of 1×10⁵ cells/ml was achieved. In a 96-well plate, 100 μl cell suspension per well was cultured for 24 h. The cells were subsequently incubated with varying concentrations of podophyllotoxin for a further 72 h; 12 wells were used per concentration. Cells incubated with only 100 μl RPMI 1640 culture medium served as a control group. MTT was dissolved in phosphate-buffered saline (PBS) to provide a solution with a final concentration of 0.5 mg/ml. After 72 h, the cell suspension was discarded and 200 μl MTT solution (0.5 mg/ml) was added to each well and incubated at 37°C for 4 h. All media were removed and 150 μl dimethyl sulfoxide was added to each well in order to dissolve the purple formazan crystals. The plate was agitated for 3 min and the spectrophotometric absorbance at 570 nm was measured using a microplate reader. The inhibitory rate and IC₅₀ was calculated.

Cells (5×10⁴/ml; 1 ml) were planted in 24-well plates and cultured for 24 h with three wells per group. Subsequently, the cells were treated with various drug concentrations for 48 h. The cells were harvested via trypsinization, washed twice with cold PBS and fixed in 70% cold ethanol for 12 h at 4°C. The fixation fluid was then discarded and the cells were washed with PBS twice. PI was added to the samples for 30 min for staining and the samples were measured by flow cytometry with an excitation wavelength of 488 nm.

Cells (5×10⁴/ml; 1 ml) were planted in 6-well plates, cultured for 24 h and treated with varying drug concentrations for 48 h. Cells were harvested via trypsinization, washed twice with cold PBS and fixed in 4% paraformaldehyde for 30 min at 4°C. The fixation fluid was then discarded and the cells were washed with PBS twice. Hoechst 33258 was added for 20 min, after which the dye was discarded and the cells were washed twice with PBS. The cells were observed using an inverted fluorescence microscope.

Cells (5×10⁴/ml; 1 ml) were planted in 6-well plates, cultured for 24 h and treated with various drug concentrations for 24 h. Cells were harvested via trypsinization, washed twice with cold PBS and loaded with Rhodamine 123 (5 μg/ml) for 30 min at 37°C. The fluorescence intensity of the MMP was observed using laser scanning confocal microscopy.

Cells (5×10⁴/ml; 1 ml) were planted in 6-well-plates, cultured for 24 h and subsequently treated with varying concentrations of podophyllotoxin for 48 h. Cytoplasm extracts were prepared with 150 μl cell lysis buffer on ice for 30 min. Following centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was collected and the protein concentration was quantified using the detergent-compatible protein assay kit. Proteins were mixed with SDS-PAGE sample loading buffer (Beyotime Institute of Biotechnology). In total, a 40-μg sample of protein was separated in a 10% polyacrylamide gel and blotted on a nitrocellulose membrane. The blots were blocked with blocking buffer for 2 h at room temperature and incubated with anti-cyt-c, anti-caspase-9 and anti-caspase-3 antibodies for 12 h at 4°C. Subsequently, the nitrocellulose membranes were washed with TBST buffer three times and incubated with AP-conjugated goat anti-mouse antibody in blocking buffer for 2 h at R.T. Then the nitrocellulose membranes were washed with TBST buffer three times and detected by BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology). The bands were then visualized and quantified using the Gel Doc XR imaging system (Bio-Rad, Hercules, CA, USA).

Data are expressed as the mean ± SD. Statistical analyses were performed using analysis of variance to compare the various groups. SPSS 15.0 software was used for statistical analysis (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The MTT assay revealed that following treatment with various concentrations of podophyllotoxin for 72 h, podophyllotoxin exhibited an inhibitory effect on the proliferation of SGC-7901 cells in a concentration-dependent manner. The optical density values of the treated groups exhibited statistically significant differences when compared with the control group (P<0.01). The IC₅₀ was 26.30 μmol/l (Fig. 1).

Following treatment with varying concentrations of podophyllotoxin for 48 h, the cell cycle changed and the number of cells in the G₀/G₁ and S phases decreased, while the number of cells in the G₂/M phase increased. The apoptotic peaks appeared gradually in a concentration-dependent manner, and the apoptosis rates were 11.06, 20.39 and 33.67% in the 13.15, 26.30 and 52.60 μmol/l podophyllotoxin groups, respectively, as shown in Table I and Fig. 2.

Under an inverted fluorescence microscope, the cells in the control group were observed to grow normally and the fluorescence in the cell nuclei was uniform. Following treatment with various concentrations of podophyllotoxin for 48 h, a high proportion of cells exhibited apoptosis-like changes, including cell detachment, cytoplasmic condensation and the appearance of apoptotic bodies, in a concentration-dependent manner (Fig. 3).

Following treatment with various concentrations of podophyllotoxin for 24 h, the fluorescence intensity of Rhodamine 123 in cells decreased, indicating that the level of the MMP had decreased. With increasing drug concentrations, the level of the MMP decreased accordingly and statistically significant differences were observed when compared with the control group (P<0.05), as shown in Table II and Fig. 4.

Following treatment with various concentrations of podophyllotoxin for 24 h, the expression levels of apoptosis-related proteins in SGC-7901 cells changed. The expression level of cyt-c increased, while the expression levels of pro-caspase-9 and pro-caspase-3 decreased in a concentration-dependent manner. Statistically significant differences were observed when compared with the control group (P<0.05), as shown in Fig. 5.