| Breast cancer is the common malignant tumor among women now. In many western country, the mobidity of breast cancer account for the first place in female tumor. Recent twenty years,.the incidence of breast cancer upgrades manifestly in our country,which is the majior cause of cancer-related deaths among women. The management of human breast cancer frequently includes radiation therapy as an important intervention, so improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2/neu growth factor receptor occurs in 25-30% of human breast cancers. HER-2/neu(c-erbB-2) protein is a member of the epidermal growth factor receptor(EGFR) family and has intracellular tyrosine kinase activity. Overexpression of HER-2/neu gene was often correlated with tumor invasion ,poor prognosis and therapeutic resistance. HER-2/neu has been regarded as index of cancer drug-resistance clinically and now becomes the new anti-tumor therapy target.Genistein (4',5,7-trihydroxyisoflavone),an important non-nutrition component presented in soybeans, exhibits multiple bilological activities resulting in anticancer effects. Genistein is a natural isoflavone phytoestrogen derived from soy products with a specific inhibitory effect on tyrosine-specific protein kinase and has no side effect. Genistein that possesses heterocyclic phenols is structural resemblance to estradiol and has mixed agonist/antagonist properties. Recently, the relationship between radiosenstivity and PTK activity of the breast cancer cells has received much attentions, these study shows that antibody to HER-2/neu receptor can significiantly inhibit the proliferation of breast cancer cells, decrease the DNA reparation compotent,and enhance the radiosenstivity of breast cacer cells with HER-2/neu overexpressing. Some clinically observation shows that the recurrences of breast cancer is rare in patient with low expression of HER-2/neu, and radiation treatment combined with antibody to HER-2/neu receptor or inhibition of PTK activity decreases significantly the mortality of patient with overexpression of HER-2/neu. Still, the mechanism of genistein increasing the radiosensetivity in HER-2/neu- overexpressing breast cancer cell lines remains unclear.Based on the above analysis, combining the progress of researches both at home and abroad,using MCF-7(ER+ , HER-2/neu low expression or nonexpression ),MDA-MB-453(ER- , HER-2/neu overexpression) Human breast cancer cells were emploied as study mode , firstly we successfully establish the HER-2/neu-overexpressing breast cancer through co-transfected with pCMV/ERBB2 plasmids and pCIneo plasmids in the MCF-7 breast cancer cells (which be named MCF-7/HER-2 cell). Then MTT,trypanblau assay, flow cytometry methods were performed to evaluate the proliferation and apoptosis effects of genistein and orγrays alone. The result demonstrated that genistein enhanced the radiosenstivity of breast cacers lines with HER-2/neu-overexpressing. In addition, RT-PCR, western blotting were performed to investigate the influence of genistein on Akt,P-Akt,p53,ERK2,HER-2,p21 expression on mRNA and protein level , and elucidated its mechanisms.The main results and conclusions were summarized as follow:1.After the MCF-7 breast cancer cells co-transfected with pCMV/ERBB2 plasmids and pCIneo plasmids (which be named MCF-7/HER-2 cell), and certificated with immunofluorescence technique,we successfully established HER-2/neu-overexpressing breast cancen cells .2. Human breast cancer cell lines MCF-7,MCF-7/HER-2,MDA-MB-453 were treated with genistein(5,10,20,40,70,100,130,160,190,220,250,280,310,340μmol/L) ,the result of MTT assay showed that genistein could inhibit the proliferation of MCF-7,MCF-7/HER-2,MDA-MB-453 cells.The inhibitive effect of genistein on MDA-MB-453 was increased along with the increasing concentration of genistein, and the IC50 was 100μmol/L. The proliferation of MCF-7/HER-2 and MCF-7 were enhanced upon a low concentration of genistein, but inhibited at a high concentration(40μmol/L and 10μmol/L, respectively).3. Human breast cancer cell lines MCF-7,MCF-7/HER-2,MDA-MB-453 were treated with genistein and or 10Gyγrays alone 24h,the result of apoptic rate showed that the apoptic rate of MCF-7/HER-2,MDA-MB-453 Human breast cancer cellsγrays group, contrast to the control group, was no significant difference(p>0.05),but genistein andγrays group added 32.63%(p<0.05),47.36%(p<0.05)rspectively. the apoptic rate of MCF-7γrays group, contrast to the control group, added 12.90%(p<0.05),however, genistein andγrays group had no significant change(p>0.05). These finding showed that HER-2/neu overexpressing breast cancer had the effect of radio-resistance, but genistein could increase the effect ofγrays inducing apoptosis in HER-2/neu-overexpressing breast cancer cells.4. Human breast cancer cell lines MCF-7,MCF-7/HER-2,MDA-MB-453 were treated with genistein and or10Gyγrays alone 24h. the result of westernblotting and RT-PCR showed that genistein did not change the expression of HER-2 and total Akt in MCF-7,MCF-7/HER-2,MDA-MB-453 cells,but significantly decreased phosphorylated Akt(P-Akt) level. Genistein increased the expression of p53, p21 in MCF-7/HER-2, and MDA-MB-453 cells , genistein increased the expression of p21, but significantly decreased ERK2 level. It suggested that genistein blocks PI3K and MAPK transduction pathway via inhibition of PTK activity of HER-2/neu may be of its mechanism of genistein enhance the radiosenstivity of HER-2/neu-overexpressing MCF-7/HER-2, MDA-MB-453 breast cancer cell lines.The observations suggest that genistein can inhibit the proliferation of MCF-7,MCF-7/HER-2,MDA-MB-453 Human breast cancer cells, the effect of genistein is in a dose-dependent manner,and increase the effect of apoptosis induced byγrays in HER-2/neu-overexpressing breast cancer cell lines. Genistein increase radio-therapeutic senstivity by blocking MAPK and PI3K pathway activiation mediated by HER-2/neu- overexoressing through inhibition of TPK activity of HER-2/neu in MDA-MB-453, MCF-7/HER-2 cell lines.
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