Regulation On The Stemness Of Breast Cancer Cell Line MDA-MB-231by Hypoxia

Objective:To study hypoxia regulating on the stemness of breast cancer cell line MDA-MB–231, the cell proliferation, apoptosis, cytotoxic, stem cell biomarkers expression(CD24-CD44+ESA+) and colony-forming ability, were detected using hypoxia model invitro.Methods:1.To mimic hypoxia conditions in vitro, MDA-MB-231cells were cultured inLeibovitz’s L-15in a humidified airtight chamber equipped with an air lock and flushedwith5%CO2/94%N2and1%O2. The chamber was then sealed and kept at37°C.2.Experimental groups: hypoxic and normoxic control group; hypoxic culturestreated by the above model, normoxic control group cultured under normoxic conditions,two groups fully synchronized;3.Cell proliferation and activity of MDA-MB-231cells were detected with CCK-8Kit.4.The cell toxicity of MDA-MB-231cells was measured by lactate dehydrogenase(LDH) assay using CytoTox96non-radioactive cytotoxicity assay Kit.5. Annexin V-FITC and PI were applied to detect cell apoptosis of MDA-MB-231cells.6.The breast cancer stem cells’ biomarkers, CD24-CD44+ESA+, of MDA-MB-231cells were detected by flow cytometry.7.Cell self-renewal capacity was further tested by colony formation assay. Results:1. Hypoxia can inhibit slightly the growth of MDA-MB-231cells, but the inhibitoryeffect is not significant when compared with control（48hours Group P>0.05）.2. Cytotoxicity assay values of hypoxia group (9.871±0.553)%, normoxic controlgroup(10.002±0.417)%;the cell toxicity of MDA-MB-231cells was not affected byhypoxia (P>0.05).3.Apoptosis rate of hypoxia group (0.97±0.74)%, normoxic groups (4.97±0.42)%;hypoxia can significantly block the cell apoptosis of MDA-MB-231cells (P<0.05).4. The rate of stem cell biomarkers expression of hypoxic groups was (3.60±0.30)%,normoxic control groups was (1.33±0.21)%; the cell proportion of CD24-CD44+ESA+inMDA-MB-231cells increased obviously after they were treated with hypoxia.;5. Clone formation assay was divided into50cells,100cells,200cells groups,normoxic groups cloning rate were (6.67±0.67)%,(6.67±0.88)%,(6.50±0.29)%respectively, hypoxia group cloning rate were (19.33±1.76)%,(17.67±1.45)%,(19.50±1.00)%respectively; cell colony formation rate of MDA-MB-231increased significantlyin hypoxia-treated cells (P values of three groups were P <0.05, P <0.05, P <0.01).Conclusion:1. Hypoxia contributes to inhibition of cell apoptosis of MDA-MB-231cells, but notinhibition of cell toxicity and cell growth.2. Hypoxia can regulate the stemness of MDA-MB-231cells, and increase their rateof cell colony formation.