The human prostate LNCaP cancer cell line was purchased from the Cell Resource Center of the Shanghai Sangon Biological Engineering Technology and Service Co. (Shanghai, China), RPMI-1640 medium and trypsin were both purchased from Gibco (Carlsbad, CA, USA), neonatal calf serum was purchased from Hyclone (Logan, UT, USA), the cell culture incubator was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

LNCaP cells were routinely thawed, cultured and passaged. For experiments the cells were divided into two batches, cells in the control group were grown in a 5% CO₂ + 20% O₂ environment and cells in the hypoxia group in a 5% CO₂ + 1% O₂ environment. All the cells were cultured at 37°C with saturated humidity. RPMI-1640 containing 10% neonatal calf serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin were used as culture media. The cells grew adhering to the flask wall and media were exchanged every two days. The cells were digested and passaged with 0.25% trypsin, and cells in the logarithmic growth phase were used for the subsequent experiments.

The percentage of CD13⁺ cells in each group was determined by flow cytometry, the expression of HIF-1α and VEGF were detected by western blot analysis, the cell proliferation rate was detected by the MTT assay and the apoptotic rate was detected by flow cytometry at 12, 24 and 72 h respectively.

Equivalent amounts of cells from each group were resuspended at final concentration of 2×10⁶/ml and mixed with mouse anti-human monoclonal antibody (1:200; Sigma-Aldrich, St. Louis, MO, USA). The mixtures were incubated for 1 h at room temperature in the dark and then centrifuged at 2,000 × g for 10 min. The supernatants were discarded. Then, 5 ml phosphate-buffered saline (PBS) (10 mM, pH=7.4) were added and the samples were resuspended, after centrifugation at 2,000 × g for 10 min, the supernatants were discarded again, 1 ml PBS was added and the samples were resuspended again. The percentages of CD133⁺ cells were determined within 1 h using a FACSCaliber flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

To determine the apoptotic cell percentages equivalent amounts of cells were centrifuged at 1,500 × g for 10 min, the supernatants were discarded and the samples were washed with PBS. After centrifugation at 1,500 × g for 10 min the precipitates were collected. Binding buffer (100 µl) and 10 µl of FITC-conjugated Annexin V (20 µg/ml) (both from Jiangsu Biyuntian Technology, Jiangsu, China) were added to each sample and were placed in an incubator for 30 min at room temperature in the dark. Subsequently, 5 µl of PI (50 µg/ml; Jiangsu Biyuntian Technology) were added and the samples were incubated for 5 min in the dark. Then, 400 µl binding buffer were added and the samples were detected within 1 h by flow cytometry. The cells without Annexin V-FITC and PI were used as negative controls.

Equivalent amounts of cell culture medium from each batch of cells were centrifuged at 1,500 × g for 10 min and the supernatants were discarded. The samples were washed with PBS and then centrifuged at 1,500 × g for 10 min, 0.2 g of cell precipitate were collected and washed with PBS. RIPA lysis buffer (500 µl; Jiangsu Biyuntian Technology) was added, each sample was homogenized in a vortex 10 times and then transferred to a centrifuge tube. The centrifuge tube was placed in an ice bath and proteins were allowed to lyse for 30 min, then each sample was centrifuged at 4°C at 3,000 × g for 5 min and the supernatant was transferred to 1.5 ml centrifuge tubes and stored at −20°C.

The BCA assay (Jiangsu Biyuntian Technology) was used to measure protein concentrations and purity. Total protein (30 µg) was mixed with 5X loading buffer. A 10% separation gel and a 4% concentration gel were prepared, and the samples were separated by 8% SDS-PAGE (Invitrogen, Carlsbad, CA, USA). The electrophoresis voltage was set at 80 V for 30 min for the concentration gel and 120 V for 3 h for the separation gel (until the bromophenol blue reached the bottom of the gel). The separated proteins were transferred to a PVDF membrane (Invitrogen). Mouse monoclonal HIF-1α antibody (dilution, 1:500; cat. no. ab113642), mouse monoclonal VEGF antibody (dilution, 1:500; cat. no. ab9530) were added and the membranes were incubated overnight at 4°C, β-actin was used as internal reference protein for normalizing quantities. Secondary rabbit anti-mouse (HRP) IgG antibody (dilution, 1:2,000; cat. no. ab6728) was added and the membranes were incubated at room temperature for 4 h,then the membranes were washed with PBS, ECL was added onto each membrane and blots were developed in the dark. Images were scanned and stored. The data was semi-quantitatively analyzed with LabWorks 4.5 Image Analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell samples were resuspended to a final concentration of 2×10⁶/ml and seeded in 96-well plates with 100 µl/well. After 12, 24 and 72 h, 10 µl of 5 mg/ml MTT (Bio-Rad Laboratories, Inc.) were added into each well. After 4 h, the supernatants were discarded and 150 µl of DMSO (Bio-Rad Laboratories, Inc.) were added into each well. The samples were agitated for 10 min and the optical density (OD) at A490 nm was measured in a microplate reader (Bio-Rad Laboratories, Inc.). Samples were measured three times, and the calculated average was used as the result in each case. The cell proliferation rates were calculated according to the following formula: Proliferation rate (%) = OD value of the experimental group/OD value of the control group × 100%.

Statistical analysis was performed using SPSS 20.0 statistical software (SPSS, Inc., Chicago, IL, USA). Measurement data were expressed as means ± standard deviation, and the one-way analysis of variance was used to compare the data at different time-points. P<0.05 was considered to indicate a statistically significant difference.

The percentage of CD133⁺ cells in the hypoxia group increased gradually from 12 to 24 to 72 h, while there was no change in the control group. The difference at each time-point was significantly higher in the hypoxia group (p<0.05) (Fig. 1).

The expression levels of HIF-1α and VEGF in the hypoxia group were gradually increased from 12 to 24 and to 72 h, while the levels in the control group exhibited no changes. The level in the hypoxia group at each time-point was significantly higher than the corresponding level in the control group (p<0.05) (Fig. 2).

The cell proliferation rates in the two groups increased gradually from 12 to 24 and to 72 h, but the proliferation in the hypoxia group was significantly higher than that in the control group (p<0.05) (Fig. 3).

There were no significant changes in terms of the apoptotic cell percentages between the two groups at different time-points, but the percentage in the hypoxia group was significantly lower than that in the control group at each same time-point (p<0.05) (Fig. 4).