JB (molecular weight of 330.4 kDa, chemical formula C₂₈H₂₆O₄, purity >99%) (Fig. 1) was kindly provided by Professor Shujun Zhang (Chemical Engineering Institute, Qiqihar University). JB was dissolved in dimethylsulfoxide (DMSO) to make a stock solution at a concentration of 100 mM, which was further diluted to the appropriate concentration with culture medium prior to each experiment. Control experiments contained DMSO.

The human breast cancer cell line MCF-7 was obtained from the Laboratory of Molecular Oncology, Beijing Cancer Hospital/Institute. Cells were incubated in complete DMEM (Gibco BRL; Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco BRL) in a humidified atmosphere at 37°C and 5% CO₂.

Cells (1×10⁴/ml) were cultured in 96-well chamber slides for 24 h prior to use in the experiment. Culture mdium was replaced with fresh medium containing the appropriate concentration of JB ranging from 0 to 80 μg/ml, and fresh medium with the drug was added from 24 to 72 h. MTT (Sigma-Aldrich; St. Louis, MO, USA) was added into each well and cultured for another 4 h, and the supernatant was discarded and supplemented with 100 μl DMSO. When the crystals were dissolved, the absorbance values were read on the enzyme-labeled mini reader II (Bio-Rad; Hercules, CA, USA) at the wave length of 570 nm. The procedure was repeated three times.

Cells were seeded in 60-mm culture dishes and grown to 50% confluence. Subsequently, the cells were cultured in serum-free medium for 24 h and then incubated with 0, 10, 20, 40 μg/ml of JB for 24 h in complete medium. The cells were harvested by trypsinization, centrifuged at 2,000 rpm for 5 min, washed in PBS, and resuspended in cold 70% ethanol. Finally, 1 ml propidium iodide stain solution (20 μg/ml PI, 100 μg/ml DNase free RNase A) was added to the samples which were analyzed on a FACScan (Becton-Dickinson; San Francisco, CA, USA) within 30 min. Data on 20,000 cells were acquired and processed using Lysis II software (Becton-Dickinson).

Cells were seeded in 6-well plates at a density of 1.2×10⁶ cells/well. After 24 h of JB treatment at a concentration of 0, 10, 20, 40 μg/ml, cells were collected, washed twice with cold PBS and 1×10⁶ cells were resuspended in 500 ml 1X binding buffer. Subsequently, 100 μl of cell suspension was transferred to a 5 ml culture tube and incubated with 10 μl of Annexin V antibodies and 10 μl of propidium iodide containing 300 μg/ml RNase (Sigma). The cells were vortexed gently and incubated for 15 min at room temperature (RT) in the dark. 1X binding buffer (400 μl) was added to each tube and the cells were analyzed with flow cytometry within 1 h.

In brief, small sections of tumor tissue (1 mm³) from control and treated mice were fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 4 h at RT (24°C). This was followed by washing the tissue samples in 0.1 M sodium phosphate buffer (pH 7.4) and then placing them in 2% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4) for 2 h at room temperature. Dehydration was carried out in an ascending grade of ethanol, followed by embedding in Epon 812 and polymerization at 60°C for 48 h. Ultrathin sections (50–70 nm) were obtained using an Ultracut Ultra microtome (Leica Microsystems GmbH; Wetzlar, Germany) and picked up onto 200-mesh copper grids. The sections were double-stained with uranyl acetate and lead citrate and then analyzed under an FEI Tecnai-12 twin transmission electron microscope equipped with an SIS Mega View II CCD camera at 80 kV (FEI Co.; Hillsboro, OR, USA).

Western blot analysis was performed as previously described. Proteins were transferred to a nitrocellulose membrane and blots were probed with anti- cyclinD1, cyclinE, p-PI3K, p-Akt, PTEN, mTOR, p-eIF4E and β-actin followed by incubation with horseradish peroxidase-conjugated mouse or rabbit immunoglobulin. Blots were then developed using the West Pico Chemiluminescent substrate (Pierce; Woburn, MA, USA).

All animal procedures were approved by the Committee on Animal Experimentation of Beijing Cancer Hospital and the procedures complied with the NIH Guide for the Care and Use of Laboratory Animals. Female Balb/c nude mice (4–5 weeks of age), obtained from the Beijing Vital River Experimental Animals Technology (Beijing, China), were used in all experiments. Suspensions of 5×10⁵ cells for MCF-7 in 100 μl PBS were subcutaneously inoculated into the right and left flank of the nude mice (n=3 in each group). JB (40 mg/kg) was administered intraperitoneally every three days for one month. Tumor growth was measured with a caliper every three days using the formula, tumor volume V (mm³) = height × length × depth. All of the nude mice were fed for one month. At the end of the experiment, tumor tissues were weighed and harvested for additional analyses as described below.

All tumor tissues were fixed with 4% paraformaldehyde (pH 7.4) and embedded in paraffin. All were plated on coverslips and fixed with cold acetone for 10 min. Following washing with PBS, coverslips were treated with methanol-hydrogen peroxide (97 ml:3 ml) for 30 min at RT. All coverslips were washed in PBS and treated with 1:100 dilution of antibody (p-PI3K, p-Akt, PTEN, mTOR, IgG conjugated with HRP) for 1 h at 37°C. Coverslips were washed thoroughly and treated with 0.05% DBA containing 0.05% fresh hydrogen peroxide. Coverslips were counter-stained with haematoxylin and observed under a light microscope.

Each experiment was performed at least three times. All data were expressed as the mean value ± standard deviation (SD). The means of the different groups were compared using one-way of variance (ANOVA). All statistical analyses were performed with the SPSS17.0 software (SPSS; Chicago, IL, USA). Differences were considered statistically significant at p<0.05, p<0.01, p<0.001. Log IC₅₀ calculation was performed using the built-in algorithms for dose-response curve with variable slope. A fixed maximum value of the dose-response curve was set to maximum obtained value for each drug.

Cell viability was measured by MTT analysis. The treatment of MCF-7 cells for 24–72 h with 0–80 μg/ml resulted in a dose-dependent decrease in cell proliferation (Fig. 2A). Following treatment with the different concentrations of JB for 24, 48, and 72 h, the growth activities of drug treatment groups were reduced in a time-dependent manner by IC₅₀ (Fig. 2B), (p<0.05, p<0.01).

The percentages of G1, S, and G2/M cells were evaluated by PI staining and flow cytometric analysis. The apoptotic rates (APO) of the experimental groups (9.09±1.13% and 11.56±2.25%) were higher than those of the untreated control group (4.21±0.54%). Together with the prolongation of time, the experimental group of JB (40 μg/ml) showed typical apoptosis peak (21.23±4.25%). Meanwhile, the cell cycle also showed notable change with the appearance of S peak and increased percentage of S cells (Fig. 3B). When cells were treated with 40 μg/ml JB for 24 h, we found a significant number of cells that was more prominently arrested at the S phase of the cell cycle (31.27±3.91%) than in the JB 0 μg/ml group (16.48±1.7%) (Fig. 3B) Furthermore, we performed phosphatidylserine externalization (Annexin V binding) and cell membrane integrity (PI staining) binding assay to detect the redistribution of phosphatidylserine, which is a hallmark for early apoptotic cells.

Annexin V-FITC/PI dual labeling further showed that after treatment with JB (10, 20 and 40 μg/ml for 24 h), the maximum concentration of JB induced a higher percentage of apoptosis cells; JB 40 μg/ml (32.61±5.87%) compared to JB 0 μg/ml (6.24±1.24%) (Fig. 3A). The representative morphological changes in MCF-7 cell were observed by transmission electron microscope (original magnification, ×10,000). The JB 0 μg/ml group of cells cultured membrane showed normal, the nuclei were round and homogeneous, the structure displayed after staining. With the increasing concentration of JB, apoptotic cells appeared more and more in the JB-treated group. The typical characteristics of apoptosis were cytoplasmic edema and nuclei condensed, the fragmented chromatin arranged against the nuclear membrane when MCF-7 cells were treated with JB (10, 20, 40 μg/ml) for 24 h. The JB 40 μg/ml group of cells presented necrosis (Fig. 3C).

To confirm the antitumor effect of JB in vivo, MCF-7 cells were transplanted into nude mice. There was no statistical difference between the four groups at the start of treatment (P>0.05), and they were randomly divided into four groups: the negative control group, the JB group (40 mg/kg), the 5-Fu group (5 mg/kg), and the JB+5-Fu group. Tumor growth curves were depicted (Fig. 4C) to compare the differences in the antitumor efficacy during the course of the experiments. At 28 days, tumor volume in the 5-Fu, the 5-Fu+JB and the JB group were greatly reduced, while tumors in the control group reached 1,207 mm³. The differences in the tumor sizes of the four groups was significant (P<0.05); however, no significant difference was observed between the JB and the JB+5-Fu group (P>0.05). The results of tumor weight were consistent with the results of tumor volume (P<0.05 vs. control group, P<0.01 vs. control group) (Fig. 4D).

The transplanted tumor tissues were dispersed into single cells for analysis by flow cytometry. Fig. 5B shows that tumor tissues from the 5-Fu, the JB and the JB+5-Fu group presented notable changes with increased percentage of S cells and decreased percentage of G2/M cells. The apoptotic rate in the JB+5-Fu group (31.26±3.15%) was higher than that in the control group (4.24±1.25%). Annexin V-FITC/PI dual labeling further showed that treatment with JB induced a higher percentage of apoptosic cells in the JB 40 mg/mg+5-Fu 5 mg/mg (31.26±6.24%) than in the control group (4.24±2.19%) (P<0.05) (Fig. 5A). Fig. 5C shows cellular morphology by TEM. The JB group showed that the nuclear DNA of apoptotic cells displayed nucleus pyknosis and distortion, chromatin condensation, margination, and fracture.

The expression of cyclinD1, cyclinE, mTOR, p-PI3K and p-Akt in the the tumor tissues of the JB group was significantly lower than that in the control group (P<0.05) (Fig. 6). This is in contrast to the upregulation of PTEN and p-eIF4E compared to that in the control group (P<0.05). Immunohistochemical staining of p-PI3K, p-Akt, PTEN, mTOR showed the expression of proteins in tumor tissues. By staining the PTEN of the JB group, its staining intensity became gradually stronger than in the control group (P<0.05), indicating high cell proliferation activity. Staining intensity of p-PI3K, mTOR and p-Akt was significantly weaker in nude mice treated with JB compared to the control group (P<0.01) (Fig. 7). These results reveal that JB was able to downregulate the expression of the p-PI3K, mTOR and p-Akt proteins whereas it upregulated the expression of the PTEN protein.