Human prostate RWPE-1 and prostate cancer PC-3 cell lines were obtained from the Animal Experiment Center of the Fourth Military Medical University (Xi’an, China), and (-)-gossypol was obtained from the College of Life Science of Xi’an Jiaotong University (Xi’an, China). Written informed consent was obtained from the patient prior to this. The polymer carrier, mPEG-Mal (5,000 D; Beijing Kaizheng Biotech Development Co., Ltd., Beijing, China), MTT dye (Shanghai Sangon Biotech Co., Ltd., Shanghai, China) and acridine orange (AO) dye (One Lambda, Beijing China) were purchased for the purpose of the experiments. Reverse transcription polymerase chain reaction (RT-PCR) primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

The following instruments were used in the experiments: NuAire AutoFlow CO₂ cell incubator (NuAire, Plymouth, MN, USA); PCR EDC-810 amplifier (Dongsheng Biotech Co., Ltd., Beijing, China); multifunctional gel imaging system (GL2200; Kodak, Rochester, NY, USA); JEM-2000EX transmission electron microscope (Electronic optical Company, Osaka, Japan); and BX60 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan).

An emulsification-volatilization method was used to prepare the loaded (-)-gossypol nanoparticles. Blank nanoparticles were also prepared using the same method, after which they were frozen. The average diameter of the nanoparticles was 65.1 nm, the (-)-gossypol-loading efficiency was 97.5±1.57% and the loading capacity was 37.5±0.27%. In vitro release experiments demonstrated that the (-)-gossypol nanoparticles had controlled-release characteristics.

PC-3 cells were adjusted to a concentration of 5×10⁶ cells/ml and inoculated in 96-well culture plates, with each well holding up to a volume of 100 μl. Free (-)-gossypol or (-)-gossypol nanoparticles at different concentrations were added to a plate (one plate for each concentration of gossypol nanoparticles), and the final concentrations in the wells were 2.5, 5, 10 or 20 μg/ml. Each well was followed by three duplicate wells. After 48 h, 20 μl MTT (5 mg/ml) was added and the plates were cultured for 4 h. The culture solution was centrifugally removed. Next, 150 μl DMSO was added to each well and the plate was vortexed for 10 min until the crystals were fully dissolved. ELISA was used to detect the absorbance (optical density) at a wavelength of 490 nm, and the median inhibitory concentration (IC₅₀) was calculated. The same method was used to measure the growth inhibition of the blank nanoparticles (control sample) on the PC-3 and RWPE-1 cells, in order to assess the toxicity of the polymer carrier.

PC-3 cells were inoculated in 96-well plates, with each well containing 100 μl cell suspension. In each well, 100-μl samples of the different (-)-gossypol nanoparticle concentrations were added. For the control group, 100 μl RPMI-1640 culture medium (ScienCell, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco^®, Invitrogen Life Technologies, Grand Island, NY, USA), was added. After 48 h, 10 μl AO dye (Bio-Teck, Beijing, China) was added to each well and cultured for 15 min. The morphological changes were observed under an inverted fluorescence microscope (Olympus Corporation). Each sample was found to contain ≥100 cells, and the percentage of apoptotic cells was calculated.

Cells were inoculated in culture bottles at a concentration of 2×10⁵ cells/ml, with each bottle containing 4 ml cell suspension. After 24 h, 4 ml (-)-gossypol nanoparticles, at a concentration of 10 μg/ml, was added to each bottle. For the control, 4 ml DMSO (Beyotime, Hangzhou, China) at the same concentration was added to the RPMI-1640 culture medium. After 48 h, trypsin was used to digest and wash any unreacted RPMI-1640 culture medium, and the solution was centrifugally subsided. Next, 4% glutaraldehyde (Huakang, Suzhou, China) was added and incubated for 2 h, which was followed by two washes with phosphate-buffered saline. Osmic acid (1%) that had been precooled at 4°C was added, and after 1 h, the samples were dehydrated, embedded in paraffin and cut into 70-nm segments. Uranyl acetate and citrate staining were used to dye the samples, and their cellular morphology was observed under a transmission electron microscope.

The concentration of the cells was adjusted to 5×10⁵ cells/ml. A total of 2 ml cells was added to a cell culture bottle. Samples from the control group and the experimental group (containing 10.0 μg/ml (-)-gossypol nanoparticles) were placed into the cell culture bottle; 8 ml cell culture fluid was added and cultured for 48 h. The primer design and the experimental methods followed in these experiments were based on the methods of a previous study (13).

SPSS 10.0 software (SPSS, Inc., Chicago, IL, USA) was used to conduct statistical analysis. χ² analysis and the t-test were used to evaluate the results, where P<0.05 was considered to a indicate statistically significant difference.

When the (-)-gossypol nanoparticles and free (-)-gossypol reached a concentration of 10.0 μg/ml, they demonstrated evident antitumor activity against prostate cancer PC-3 cells in vitro (Fig. 1). As shown in Fig. 1, the inhibition effects of (-)-gossypol nanoparticles and free (-)-gossypol on the proliferation of PC-3 cells increased with time. In addition, following culture for 72 h, the inhibition effects of (-)-gossypol nanoparticles and free (-)-gossypol on the proliferation of PC-3 cells increased with increasing concentration (Fig. 2). At the various time points, the IC₅₀ of the (-)-gossypol nanoparticles was slightly higher compared with the free (-)-gossypol; however, no statistically significant difference was observed (P>0.05).

Following the addition of the blank carrier in the PC-3 and RWPE-1 cells for 48 h, no evident change was observed with regard to the survival rate of the cells. When the concentration of the blank carrier reached 200 μg/ml, the survival rate of the PC-3 and RWPE-1 cells decreased; however, the rate remained >95%, and no evident change in the cell shape was observed (data not shown).

After culturing the control group cells for 48 h, the cells were concentrated together and the cell chromatin was evenly distributed (Fig. 3). Upon culturing with 5.0 μg/ml (-)-gossypol nanoparticles for 48 h, only part of the cell nucleus was pyknotic and cell apoptosis was observed. Following culture with 10.0 μg/ml (-)-gossypol nanoparticles for 48 h, the number of apoptotic cells was markedly increased, the cell chromatin was not evenly distributed and a number of cells had burst. After culturing with 20.0 μg/ml (-)-gossypol nanoparticles for 48 h, the number of cells decreased, cell apoptosis was evident, the chromatin was arranged along the nuclear membrane in a crescent-shape and cell fragmentation was observed.

In the normal PC-3 cells, microvilli were detected on the surface, small fat droplets and lipofuscin particles were observed, and the nucleus chromatin was shown to mainly consist of euchromatin (Fig. 4A). Following culture with 10.0 μg/ml (-)-gossypol for 48 h, the PC-3 cells presented typical features of apoptotic cells, including the disappearance of microvilli from the cell surface, smooth edges and agglutinated nuclear chromatin that was arranged close to the edge of the nuclear membrane (Fig. 4B).

Through semi-quantitative RT-PCR detection, the size of the Bcl-2, Bak and GAPDH genes were determined as 387, 360 and 142 bp, respectively, consistent with the expected values. Following culture with 10.0 μg/ml (-)-gossypol nanoparticles for 48 h, the mRNA expression levels of Bcl-2 were downregulated in the PC-3 cells, and the Bcl-2/GAPDH ratio decreased from 0.17 to 0.08. In addition, the mRNA expression levels of Bak were upregulated, and the Bak/GAPDH ratio increased from 0.62 to 0.89.