The Effect Of Evn-50 On The Growth And Apoptosis Of Human Breast Cancer MCF-7 Cell Line With Tamoxifen-resistance

Purpose:Breast cancer is the most common malignancy among women, affecting women's health and quality of life seriously. Tamoxifen can increase the disease-free survival and overall survival in the breast cancer patients of estrogen receptor-positive, and reduce local and ipsilateral recurrence, so it is widely used in the clinical practice. In recent years, the phenomenon of tamoxifen resistance has severely limited its effectiveness, which is the clinical major problems demanding prompt solution. Study demonstrated that the ethyl acetate extract Evn-50, derived from the fruits of Vitex negundo, has broad-spectrum anti-tumor activities, including breast cancer cells.It can inhibit a variety of tumor cells growth and induce apoptosis. This experiment of Evn-50 in vitro on human breast cancer MCF-7 and MCF-7/TAM-R cell growth and apoptosis, explore to the affect and mechanisms of Evn-50 on the tamoxifen-resistant breast cancer cells, and provide a preliminary basis for further experimental studies and clinical applications in the future.Methods:The MCF-7 cell line was originally obtained from the ATCC Bank. The establishment of the antiestrogen-resistant cell lines MCF-7/TAM-R were generated by long-term treatment of MCF-7 cells with 5×10-6 M tamoxifen. Each cell line was divided into four subgroups, and treated by DMSO,5uM TAM,5uM TAM+50ug/ml Evn-50 and 50ug/ml Evn-50. Cell viaility and survival (%) was measured using a cell proliferation kit (MTT). The arrest of cell cycle in G2/M (%) and the influence induce cell line apoptosis after they treated by TAM, Evn-50 and Evn-50 combined with TAM was detected by PI staining flow cytometry. Using the method of Western blotting observe the mechanism of Evn-50 on the growth and apoptosis of tamoxifen-resistant cell line.Results:Compared with MCF-7/TAM-R cells, normal MCF-7 cells emerged many cell debris in microscopy after treated by TAM. In TAM group, Evn-50 or Evn-50 combined TAM group, the cell viaility of MCF-7 decreased more than control group (*p<0.05,**p<0.01). And in Evn-50 or Evn-50 combined with TAM group, the cell viaility of MCF-7/TAM-R decreased more than control group or TAM group, (*p<0.05, **p<0.01). In EvnO-50 or Evn-50 combined TAM group, the index of apoptosis increased more than TAM group (**p<0.01). The effects of apoptosis were dependent on the time after treated by Evn-50 or Evn-50 combined with TAM. In 72h, the effects of apoptosis and the proportion of G2/M phase cells had the most obvious significant difference (**p<0.01). After treating by TAM and Evn-50 alone or combination in MCF-7 cells, Western Blot results suggested that the expression of phosphorylated AKT (Ser473) and p-MAPK44/42 decreased, while the expression of total AKT (Ser473) and p-MAPK 44/42 were stable. It hinted that the inhuibiting effect of Evn-50 on growth of breast cancer MCF-7 cells may be can attributed to the downregulation of AKT and MAPK signaling pathway. In MCF-7/TAM-R cells, the expression of phosphorylation of AKT (Ser473) and p-MAPK44/42 protein have not changed in Evn-50 or TAM alone group, but in Evn-50 combined with TAM group, they showed reduction of the expression of the phosphrylation AKT (Ser473) and p-MAPK44/42 protein.Conclusion:Evn-50 possess the effects of the growth inhibition and apoptosis induction in MCF-7 and MCF-7/TAM-R cell lines. Particularly, can enhance TAM to induce MCF-7/TAM-R cell lines apoptosis. The mechanisms are not fully understood, may be related to the downregulation of AKT and MAPK signal pathways. It needs further experimental studies to confirm, and it is our next research focus.