| Objective: Two kinds of human breast cancer cell lines were selected for our studywith different invasive abilities(MCF-7, a estrogen dependent cell line with lowinvasive ability and MDAMB-231, which is estrogen independent with high invasiveability). The expression of erythropoietin(EPO) and erythropoietin receptor(EPO-R)in both MCF-7and MDAMB-231cells were measured from mRNA and proteinlevels. MCF-7and MDA-MB-231cell lines were treated with rh-EPO of suitableconcentration with the purpose of estimating the effects of rh-EPO on the biologicalbehavior such as cell proliferation, apoptosis, differentiation and metastatic activitiesand revealing its function role in breast cancer.The inflammatory mediator COX-2inboth two cell lines were observed after treated with rh-EPO in terms of an effectivedose and time point so as to determine whether COX-2is mediated EPO promotingcell proliferation and invasion.Methods:1. The expressions of EPO and EPO-R were determined by reversetranscription-polymerase chain reaction (RT-PCR), Western Blot andImmunocytochemistry (ICC).2. The proliferation abilities of MCF-7andMDA-MB-231cells interfered by rh-EPO in different concentration were detected byMTT assay; The apoptosis of MCF-7and MDAMB-231cells were investigated byterminal deoxynucleotidyl transferase mediated dutp-Biotin nick end-labeling assay(TUNEL) and Annexin V-FITC/PI fluorescence stain; The migration and invasionabilities of MCF-7and MDA-MB-231cells interfered by rh-EPO in differentconcentration for24h and48h were investigated by transwell technique.3. Theinflammatory mediators COX-2were detected by Enzyme Linked ImmunosorbentAssay (ELISA) to study the mechanism of EPO/EPO-R.Results:1. Western Blot、RT-PCR and ICC analysis demonstrated that MCF-7andMDA-231expressed EPO and EPO-R proteins on the cytoplasm and plasmalemma. 2. rh-EPO could promote the proliferation of MCF-7and MDA-MB-231cells ina dose and time-dependent manner and the effecting on MCF-7cells is better than onMDA-MB-231cells.3. In AnnexinV-FITC/PI fluorescence stain, the AI of the control group,200U/ml,300U/ml,400U/ml experimental groups in MCF-7cells were (6.72±2.81)%,(7.17±2.36)%,(4.15±1.86),(6.58±1.28)%respectively. There was no statisticalsignificance in these groups(F=1.192，P=0.373). The AI of the control group,200U/ml,300U/ml,400U/ml experimental groups in MDA-MB-231cells were(34.54±2.77)%(32.45±1.46)%,(30.99±4.01)%,(29.80±3.44)%respectively. There was nostatistical significance in these groups (F=1.325, P=0.412).In TUNEL, the AI of control group,200U/ml,300U/ml,400U/ml experimentalgroups in MCF-7cells were (8.07±0.118)%,(7.48±0.77)%,(7.07±0.90)%,(6.93±0.38)%respectively. There was no statistical significance in these groups(F=2.204， P=0.189). The AI of control group,200U/ml,300U/ml,400U/mlexperimental groups in MDA-MB-231cells were (31.97±0.68)%,(31.32±0.886)%,(30.82±1.95)%,(29.90±2.26)%respectively. There was no statistical significance inthese groups (F=0.894, P=0.485). It is show rh-EPO did not inhibit cell apoptosis.4. The migration cells of MCF-7were (23.00±2.12),(27.60±2.07),(43.75±5.31)respectively in the200U/ml,300U/ml,400U/ml experimental groups after24h, morethan that of the control group(6.60±1.51). The differerce between these groups wassignificant (F=121.662, P=0.000). The migration cells of MDA-MB-231were(130.50±4.93),(231.50±2.38),(347.75±3.20) respectively in the200U/ml,300U/ml,400U/ml experimental groups, in contrast to (62.60±1.52) in control group after24h.The differerce between these groups was significant (F=6715.455, P=0.000).The invasion assay revealed that the MCF-7invading cells were (18.60±3.91),(26.20±1.30),(42.25±5.68) in the200U/ml,300U/ml,400U/ml experimentalgroups,in contrast to (5.60±1.14) in control group after48h. The differerce betweenthese groups was significant (F=92.103, P=0.000). The MDA-MB-231invading cellswere (119.80±5.81),(206.80±4.32),(295.00±20.82) in the200U/ml,300U/ml,400U/ml experimental groups, and the control group only was (55.75±1.95). These group had statistical significance (F=378.139, P=0.000).The number of migration and invasion of MCF-7and MDA-MB-231cells wereincreased in dose-dependent.6. rh-EPO could increase the expression of COX-2of MCF-7and MDA-MB-231cells in a dose and time-dependent manner. And the expression of COX-2in MCF-7was higher than MDA-MB-231in the same condition.Conclutions1. MCF-7and MDA-MB-231had a low expression level of EPO and EPO-R.rh-EPO could enhance the capacity of proliferation and the ability of migration andMatrigel-invasion in vitro in both MCF-7and MDA-MB-231cell lines in a time anddose-dependent manner, but could not induce cell apoptosis。2. COX-2could play mediated role in this phenomenon that rh-EPO promotedthe proliferation and invasion of MCF-7and MDA-MB-231cell lines. The differentexpression level of COX-2in the two kinds of cells could be one reason of causingthe different proliferation effecting... |
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