Establishment and comparative analysis of 2 hypoxia models of human breast cancer MDA-MB-231 cells |
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Authors: | FANG Tian-xing NI Yu-fang LAI De-ping ZENG Fan-cai |
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Affiliation: | 1.Laboratory of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southwest Medical University, Luzhou 646000, China;2.School of Public Health, Southwest Medical University, Luzhou 646000, China |
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Abstract: | AIM: To explore the differences in the effects of 2 representative hypoxia models on human breast cancer MDA-MB-231 cells. METHODS: To establish a chemical hypoxia model, MDA-MB-231 cells were treated with CoCl2 at different concentrations (0~300 μmol/L) for different time (24 h, 48 h and 72 h). On the other hand, MDA-MB-231 cells were exposed to hypoxia with different volume fractions (1%, 2% and 5%) of oxygen for different time (4 h, 16 h and 24 h) to establish a physical hypoxia model. The viability of the cells in the 2 hypoxia models was measured by CCK-8 assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Bcl-2 and matrix metalloproteinase-2 (MMP-2) were determined by Western blot. The apoptosis of the cells was analyzed by flow cytometry. The cell invasion and migration were examined by Transwell assay and wound-healing assay, respectively. RESULTS: The HIF-1α was expressed in time- and dose-dependent manners. These results indicated that treatment of MDA-MB-231 cells with CoCl2 at 100 μmol/L for 24 h served as the final chemical hypoxia model, and exposure of MDA-MB-231 cells to hypoxia with 2% oxygen for 24 h served as the final physical hypoxia model. Compared with the cells with chemical hypoxia, the protein le-vel of HIF-1α was significantly increased in the cells with physical hypoxia. In addition, reoxygenation from hypoxia caused a rapidly degraded HIF-1α expression level in physical hypoxia model. Flow cytometry results showed that apoptosis was increased and the protein expression level of anti-apoptotic Bcl-2 was decreased in the cells with physical hypoxia, while no significant change was observed in the cells with chemical hypoxia. The protein expression level of MMP-2 in both hypoxia models was increased, and the invasion and migration capabilities of the cells with chemical hypoxia were enhanced. CONCLUSION: Two hypoxia models were successfully constructed. There were differences in the expression and stability of HIF-1α protein, and the viability, apoptosis, invasion and migration of the cells in the 2 different hypoxia models, which provide references for the selection of hypoxia models and the further study of characteristics of tumor cells in hypoxia environment. |
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Keywords: | Hypoxia Hypoxia-inducible factor-1α Apoptosis Cell viability Breast cancer |
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