MDA-MB-231 (human breast cancer cell line), KB (nasopharyngeal carcinoma cell line), U937 (human monoblast leukemia cell line), and K562 (human leukemia cell line) were purchased from the CBCAS (Cell Bank of the Chinese Academic of Sciences, Shanghai, China). Cells were maintained in RPMI1640 (GIBCO), supplemented with 10% (v/v) fetal bovine serum (HyClone), sodium bicarbonate, 100 μg/ml streptomycin and 100 U/ml penicillin (HyClone) at 37°C, in a humidified 5% CO2 atmosphere.
Antibodies and reagents
PTS2 and PT were purchased from J&K Chemical LTD. Adriamycin (ADM) was purchased from Hisun (Zhejiang Hisun Pharceutical Co., LTD). Cisplatin (DDP) was purchased from QiLu (QiLu Pharmaceutical Co., LTD.). MTT, Hoechst33258, DAPI and propidium iodide (PI) were from Sigma (Sigma Chemical Co., St Louis, MO). Antibodies to caspase-3, PARP, CyclinD1, CyclinE1 and caspase-9 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All chemicals and drugs were prepared in PBS immediately before use.
Western blotting was performed as described previously. Cells were washed twice with ice-cold PBS (pH 7.4) and lysed in a lysis buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5 mM dithiothreitol, 1 mM EDTA, 1% NP-40, 10% (v/v) glycerol, 50 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 1 mM Na3VO4. After incubation on ice for 20 min, lysates were centrifuged at 15,000× g for 10 min at 4°C and the supernatant was transferred to a clean microfuge tube. Equal amounts of the soluble protein were denatured in SDS, electrophoresed on SDS-polyacrylamide gel, and transferred to a PVDF membrane. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies were used against respective primary antibodies. Proteins were visualized using Lumi-Light Western Blotting Substrate (Roche Molecular Biochemicals). The total density of the protein bands was calculated using the Scion Image software program (Scion Corp., Frederick, MD).
Cell proliferation assay
Cells were seeded into 96-well plates at 5 × 103 cells per well 24 h before treatment. After treatment with different drugs, cell proliferation was determined using MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, 15 μl (5 mg/ml) MTT solution was added to each well, and incubated at 37°C for 4 h, after which the MTT solution was removed and 200 μl of dimethylsulfoxide (DMSO) added to dissolve the crystals. Absorbance of each well was measured at 570 nm using an ELx 800 Universal Microplate Reader (Bio-Tek, Inc.) according to manufacturer’s instructions.
Trypan blue assay
Cells were seeded in 6-well culture plates. After 24 h, culture medium containing 2.5 μg/ml of PTS2 was added to the wells. Cells were harvested at indicated times and washed with PBS, followed by centrifugation at 2500 g for 5 min. The cell pellet was then re-suspended in 1 ml of fresh culture medium; 10 μl of the cell suspension was stained with an equal volume of trypan blue (Sigma, Germany) and incubated for 2 min at 37°C. The total number of viable cells was estimated using a hemocytometer chamber.
A staining solution of Hoechst33258 was prepared immediately before use. After drug treatment, cells were collected and fixed in acetic acid/methanol (1:3) solution for 5 min at 4°C, washed 3 times with PBS, and then incubated with Hoechst33258 (50 ng /ml) for 5 min and washed 3 times with PBS. Cells were then assessed for Hoechst fluorescence in a Nikon Optiphot fluorescence microscope (magnification: ×400).
Cells for 4′-6-diamidino-2-phenylindole (DAPI) staining underwent the same PTS2 treatment as those stained with Hoechst33258. Collected cells were fixed with acetic acid/methanol (1:3) solution for 10 min at room temperature and then incubated in DAPI (1 μg/ml) for 5 min. After being washed 3 times with PBS, cells were examined using a Nikon Optiphot fluorescence microscope (magnification: ×400).
Cell cycle analysis
Cell cycle distribution was analyzed by flow cytometry. Control and treated cells were harvested, washed twice with PBS, and fixed in 70% ethanol overnight at -20°C. Fixed cells were washed twice with PBS, incubated with 1 ml of PBS containing 50 μg/ml propidium iodide, 100 μg/ml RNase A and 0.1% Triton X-100 for 30 min at 37°C. Stained cells were analyzed using a FAScan laser flow cytometer (Becton Dickinson) and ModFit LT cell cycle analysis software (Verity Software).
Apoptosis was determined using Annexin V-FITC/ PI double staining. After treatment, floating and adherent cells were collected, washed twice with PBS (pH 7.4), resuspended in 150 μl of Annexin-binding buffer and incubated with 0.4 μl of Annexin V-FITC. After 20 min incubation in the dark at room temperature, 150 μl of Annexin-binding buffer with 3 μl of PI (50 μg/ml) was added just before flow cytometry. Data were analyzed by flow cytometry using the FACSCalibur and Cell Quest software (Becton Dickinson).
Animals and solid tumor models
All experiments followed the recommendations of the Chinese Experimental Animals Administration Legislation, as approved by the Science and Technology Department of Jiangsu Province. Male ICR mice (6 weeks old, 18–20 g) purchased from Shanghai Laboratory Animal Center, Chinese Academy Sciences, were kept in groups of five animals per cage in a temperature-controlled room at 20 ± 2°C. They were fed a standard pellet diet and water ad libitum. As described in, two groups of 40 animals each were transplanted subcutaneously with hepatoma 22 (H22) tumor cells (5 × 106 cells/ml) in 0.2 ml PBS into their right groins. At 24 h after tumor inoculation, each set of 40 mice was randomly divided into 4 groups (10 mice per group) and injected intraperitoneally with PTS2 (0.25 or 2.5 mg/kg/day), DDP (25 mg/kg/day) or 0.2 ml 0.9% saline for a further 10 days. On day 11, all the animals were killed, and the tumors were dissected and weighed. Tumor growth inhibition was calculated using the formula:% inhibition = 100 × ([C - T]/C), where C is the average tumor weight of the control group and T is the average tumor weight of each treated group.
Statistical analysis used SPSS 12.0 (SPSS, Chicago, IL, USA). Results are expressed as means ± S.D. Differences between means were determined by one-way ANOVA, followed by Student–Newman–Keuls tests for multiple comparisons and Student’s t test for other data. P < 0.05 was considered statistically significant.