The breast cancer cell line MCF-7 was purchased from Shanghai Bo Valley Biological Technology Co., Ltd. (Shanghai, China), and incubated in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C with 5% CO₂. CoCl₂ was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). MCF-7 cells in the exponential phase were used for further detection. Different CoCl₂ concentrations were added to DMEM. Cells were incubated with 50, 100, 150 and 200 µM CoCl₂ for different periods of time [0, 24, 48 and 72 h for MTT assay; 24 h for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay], with an equivalent volume of PBS added to the control group. The morphological changes of MCF-7 cells were observed using an inverted phase-contrast microscope following treatment with CoCl₂.

MCF-7 cells (1×10³) were seeded in 96-well plates and cultured overnight. Subsequently, the culture medium was removed and fresh DMEM containing the aforementioned concentrations of CoCl₂ was added. Cells were then incubated for different periods of time, and the culture medium was removed and replaced with fresh DMEM with different concentrations of CoCl₂ as previously used. MTT solution (5 mg/ml, 10 µl) was added to each well prior to incubation for 4 h. Next, culture medium was removed and 100 µl dimethylsulfoxide was added to dissolve the formazan crystals. The absorbance value was determined at 490 nm.

RT-qPCR was performed to quantitatively estimate the changes in the expression of HIF-1α, CXCR4 and VEGF mRNA in MCF-7 cells treated with the aforementioned CoCl₂ concentrations for 24 h. Total RNA was isolated from cells using an RNeasy kit (Sigma-Aldrich; Merck KGaA). The RNA was reverse-transcribed into cDNA using the PrimeScript^® First Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) at 42°C for 15 min, then at 85°C for 5 min. The Maxima^® SYBR Green qPCR Master Mix (2X) kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to perform qPCR. Primer sequences for β-actin, HIF-1α, CXCR4 and VEGF are presented in Table I (13,14). The following PCR conditions were used: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec (annealing and extension, respectively). The experiment was performed in triplicate and independently repeated at least twice. RT-qPCR data were normalized and quantified using the 2^−ΔΔCq method (15). The relative expression level of HIF-1α, CXCR4 and VEGF mRNA was calculated by determining the ratio between the amount of the gene and β-actin.

The protein expression of HIF-1α, CXCR4 and VEGF was assessed by western blotting. MCF-7 cells were treated with the aforementioned CoCl₂ concentrations for 24 h and cells were lysed with ice-cold radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% NP-40, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl, pH 8.0) and protease inhibitors (AEBSF at 2 mM, Aprotinin at 0.3 µM, Bestatin at 116 µM, E-64 at 14 µM, Leupeptin at 1 µM and EDTA at 1 mM; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein concentrations were quantified using a Bicinchoninic Acid Protein assay kit. Proteins (30 µg/lane) were separated by SDS-PAGE (10% gels) and transferred onto nitrocellulose membranes. Membranes were blocked for 1 h with 5% non-fat dried milk in Tris-buffered saline containing 20% Tween-20 (TBST) and incubated overnight at 4°C with the primary antibody Following washing with TBST, membranes were incubated for 1 h with secondary antibodies. Labeled protein bands were detected using the enhanced chemiluminescence method (ProteinTech Group, Inc., Chicago, IL, USA). Western blotting was performed three times. The relative expression level of HIF-1α, CXCR4 and VEGF proteins was quantified by densitometry analysis using ImageJ (version 1.6.0; National Institutes of Health, Bethesda, MD, USA) relative to β-actin. The antibodies were as follows: Anti-HIF-1α (ab69836; 1:600), anti-CXCR4 (ab124824; 1:1,000) and anti-β-actin (ab8226; 1:1,000) were purchased from Abcam (Cambridge, MA, USA); anti-VEGF (AF5131, 1:1,000) was purchased from Affinity Biosciences (Cincinnati, OH, USA), and the secondary antibodies were purchased from ProteinTech Group, Inc.

In situ detection of apoptosis was performed on sides using the TUNEL technique using an In Situ Cell Death Detection kit (Roche Applied Science, Penzberg, Germany). MCF-7 cells were seeded onto sterile glass cover slips in a 6-well plate, and incubated with 150 µM CoCl₂ for 48 h. Cells were then assessed by a TUNEL assay, according to the manufacturer's protocol. Steps were as follows: Cells were fixed with 4% paraformaldehyde (pH 7.4, Beijing Solarbio Science& Technology Co., Ltd.) at room temperature for 1 h and washed with PBS for 5 min; cells were incubated in Blocking solution (3% H₂O₂ in methanol) for 10 min at room temperature and washed with PBS for 5 min. Cells were then incubated with penetrating fluid (0.1% Triton X-100 in 0.1% sodium citrate solution) on ice for 2 min and then 50 µl TUNEL reaction mixture (1:9) was added prior to incubation in a damp box at 37°C for 1 h. Following washing with PBS for 5 min, a drop of PBS was added to the slide. For staining of the nuclei, cells were washed with PBS and incubated with 1.0 µg/ml DAPI (Sigma-Aldrich; Merck KGaA). The samples were analyzed using a fluorescence microscope (magnification, ×100). A total of 10 fields of view were analyzed.

SPSS statistical software (version 19.0; IBM Corp., Armonk, NY, USA) to analyze the experimental data. Quantitative data are presented as the mean ± standard deviation. One-way analysis of variance with Least Significant Difference post-hoc test was used to compare between groups. Student's t-test was used to determine the significance for all pairwise comparisons of interest. P<0.05 was considered to indicate a statistically significant difference.

To examine whether hypoxia affected the mRNA expression level of HIF-1α, CXCR4 and VEGF, RT-qPCR was used to quantify mRNA levels in MCF-7 cells cultured for 48 h with different CoCl₂ concentrations. There was no significant change in the expression of HIF-1α mRNA following treatment with different CoCl₂ concentrations (Fig. 1A). However, the expression of CXCR4 mRNA and VEGF mRNA was significantly increased by treatment with CoCl₂ (50, 100, 150 and 200 µM). The 150 µM concentration of CoCl₂ induced the maximum effect (P<0.05; Fig. 1A), which indicated that hypoxia is able to promote the expression of CXCR4 and VEGF mRNA.

To examine further whether hypoxia affects the protein expression of HIF-1α, CXCR4 and VEGF, levels were examined by western blotting in MCF-7 cells cultured for 48 h with different CoCl₂ concentrations. The expression of HIF-1α protein significantly increased following treatment with CoCl₂ (150 µM; P<0.05; Fig. 1B). Expression of CXCR4 and VEGF protein was also significantly increased by CoCl₂ treatment (P<0.05; Fig. 1B). These results indicated that hypoxia also promotes the expression of HIF-1α, CXCR4 and VEGF protein. Owing to these results, a concentration of 150 µM CoCl₂ was used for further experiments.

First, in order to determine the effect of CoCl₂ on MCF-7 cell morphology, 150 µM CoCl₂ was added to MCF-7 cells for 48 h. As shown, the MCF-7 cell morphology did not change significantly after treatment for 24 h. However, 48 h later, an accumulation of cell metabolites was observed in the culture dish, and a number of the cells exhibited plasmatorrhexis (Fig. 2A). These results indicated that the high intensity of the hypoxia microenvironment may have an effect on cell morphology. A TUNEL assay was performed to investigate whether CoCl₂ was able to trigger apoptosis of MCF-7 cells. As presented in Fig. 2B, the results of the TUNEL assay demonstrated that incubation with 150 µM CoCl₂ for 48 h induced MCF-7 cells to exhibit a significant increase (5±2% TUNEL-positive cells in the control group vs. 30±5% of TUNEL-positive cells in the CoCl₂-treatment group; P<0.05) in TUNEL-positive cells, which indicated that CoCl₂ is involved in the regulation of apoptosis in MCF-7 cells.

An MTT assay was performed to determine whether CoCl₂ affects the proliferation of MCF-7 cells. The MTT assay demonstrated that MCF-7 cell proliferation significantly decreased following treatment with CoCl₂ compared with the control group (P<0.05; Fig. 3). The effects of different CoCl₂ concentrations on MCF-7 cell proliferation were as follows: The higher the concentration of CoCl₂, the higher the inhibition efficiency and the longer the treatment time, the higher the inhibition efficiency (Fig. 3). The inhibitory effect of CoCl₂ on MCF-7 cell proliferation was therefore dependent on the treatment time periods and on CoCl₂ concentration, which indicated that the time and intensity of hypoxia was able to inhibit the proliferation of cells to various extents.