Effects Of Hypoxia On The Proliferation And Its Regulating Angiogenesis-related Factors In Human Breast Cancer MCF-7cells In Vitro

Objective:To investigate the effect of hypoxic stimulation on the proliferation of humanbreast cancer MCF-7cells and on the tumor angiogenesis factors, by applying CobaltChloride（CoC12） to construct in vitro the cells hypoxia model.Methods:Human breast cancer MCF-7cells with exponential growth in routine culturewere exposed to （50,100,150,200μmol/L） CoCl₂to mimic hypoxic conditions. At12h,24h and48h, the cells were collected for morphological observation, MTT assay,RT-PCR for HIF-1α、CXCR4and VEGF mRNAs expression, The expression ofHIF-1α、CXCR4and VEGF proteins were analyzed by Western blot.Results:MTT assay showed that that CoC12inhibited the growth of MCF-7cells, in adose-and time-dependent manner. CoC12（50,100,150,200μmol/L） for24hsuppressed MCF-7cells growth by （54.62±0.07）%,（65.21±0.03）%,（80.15±0.01）%,（94.51±0.01）%, and the IC50was143.28μmol/L. Compared with the cells withoutCoCl₂treatment, the cells with CoCl₂exposure exhibited obvious morphologicalchanges and a significant growth inhibition which increased with CoCl₂concentrationand exposure time（P<0.05）, such as nuclear fragmentation, nuclear karyopycnosis,edge set of nuclear chromosomes, and the mimicked hypoxic conditions do not causedifferentiation of MCF-7cells.There were singnificantly difference between MCF-7cells treated with （50,100,150,200μmol/L） CoCl₂and the normal MCF-7cells cultrued with medium for24h in the mean relative density of over-expression ofCXCR4mRNA （P<0.05）, The relative density were0.710±0.130,0.830±0.140,0.920±0.170,0.810±0.120and0.650±0.220, respectively.the relative density of VEGFmRNA were0.490±0.110,0.630±0.140,0.800±0.090,0.430±0.140and0.55±0.060,respectively. the mean relative density of expression of HIF-1α mRNA（P>0.05）, notstatistically significant.Western blot showed that over-expression of HIF-1α、CXCR4and VEGF proteins（P<0.05）, which could be measured under hypoxia induced by （50,100,150,200μmol/L） CoCl₂for24h, The relative density of HIF-1α protein were1.000±0.061,2.730±0.150,4.810±0.420,8.170±0.520and6.890±0.380, respectively.The relative density of CXCR4protein were1.000±0.120,1.750±0.180,2.770±0.620,7.120±0.340and5.120±0.180, respectively. and the relative density of VEGF proteinwere1.000±0.010,1.450±0.071,2.670±0.170,5.350±0.150and5.650±0.150,respectively. RT-PCR and Western blot showed that hypoxia can induce theover-expression of HIF-1α and its target gene CXCR4、VEGF of human breast cancerMCF-7cells in vitro, which could regulate tumor angiogenesis.Conclusions:Hypoxia mimiced by CoCl₂exposure significantly inhibits the proliferation ofMCF-7cells, and at the non-toxic doses, CoCl₂dose-and time-dependently increasesthe expression of HIF-1α、CXCR4and VEGF.