The Effect Of X-Ray On MCF-10A EGFR Signaling Pathway And Mechanism

Objectives:Today,radiotherapy is a majority adjuvant therapeutic option for breast cancer patients,radiation can kill the cancer cells in the primary as well as metastases.Patients who accepted breast-conserving surgery and patients who had 3 or more than 3 positive axillary lymph nodes should accept the radiotherapy.Through this effective treatment,patients could gain a relatively lower local recurrence rate and get a better overall survival.Recurrence of breast cancer after radiotherapy may be partly explained by the presence of radioresistant cells.Thus,it would be desirable to develop an effective therapy against radioresistant cells.The effect of radiotherapy on DNA damage and cell phenotype may play an important role in cell radioresistance.However,there is still a reason for optimism due to recent advancement made in cancer biology which unrevealed many new deregulated pathways in cancer and their response towards drug and radiation.At the same time,whether radiotherapy will stimulate normal epidermal cells into malignant tumor cells and cause secondary tumor or not is still controversial.Research on mechanism of breast cancer radioresistance of biology and find out the therapeutic target is a key method to improve the efficacy of radiotherapy.In the treatment of lung cancer,patients with EGFR mutations accepted the tyrosine kinase inhibitor(TKI)drugs combined radiotherapy,achieved good clinical effect.Recurrence of breast cancer patients afte r radiotherapy,whether there is a certain relationship with the EGFR pathway or not,and what role had radiotherapy play on the change of the EGFR pathway,and whether application of TKI drugs in patients with breast cancer can also increase the sensitivity of radiotherapy needs further research.Method:1.By repeating low dose of X-ray irradiation to establish radiation resistance breast cancer cell lines,including MCF-7,T47 D,MDA-MB-231 and immortalized normal mammary epithelial cell line MCF-10 A2.Make use of Nikon inverted microscope(TE2000-u)to observe the changes of morphology of the cells after irradiation.3.By CCK 8 proliferation test,EDU cell proliferation experiment to study the change of radioresistanc cell proliferation and its application of epidermal growth factor(EGF),TKI and EGFR phosphorylation inhibitors AG1478 effects on cell proliferation.4.By cells scratch test,Transwell experiment to study the change of radiation tolerance ability of cell migration,and epidermal growth factor(EGF),TKI drugs and EGFR phosphorylation inhibitors AG1478 effects on cell migration ability.5.Through cell flow to test radiation influence on cell cycle,and the effect of TKI and EGFR phosphorylation inhibitors AG1478 on cell cycle.6.By Western Blotting technology to demonstrate radiation resistance cell line of the expression of EGFR and its changes of the downstream signaling pathways.Results:1.Cell lines: successfully establish radioresistance cell line MCF-10A-10 Gy.2.Cell morphological changes: mesenchymal cells development trend,the shape of the MCF-10A-10 Gy became thinner and longer cells increased pseudopodia and rising rates of the nucleus of cells;Cells between contact less,with the increase of the distance between the cells and cells are uniform,no longer assume the cluster cell growth.Don’t need EGF cell growth.3.Changes in cell biology: MCF-10A-10 Gy slow in cell proliferation,migration,application of TKI kind of small molecular drugs and EGFR phosphorylation inhibitors AG1478 processing,slow in cell proliferation,migration ability.4.The change of cell cycle: after irradiation,increase in the number of proportion of cells in G1 phase,the application of TKI kind of small molecular drugs and EGFR phosphorylation inhibitors AG1478 processing primitive cells MCF-10 A,increase proportion of cells in G1 phase.5.EGFR downstream pathways and E-cad expression changes and their relations: abnormal cells after irradiation EGFR downstream Akt pathway activation;E-cad cut,and negative correlation with Akt activation.EGF stimulation,original MCF-10 A EGFR downstream pathway activation,down regulate the expression of E-cad.Conclusion:1.X-Ray may cause mammary epithelial cell MCF-10 A the abnormal activation of the EGFR-Akt signaling pathway and its growth no longer depends on Exogenous EGF2.After the irradiation,the morphology of the MCF-10 A was significantly changed,the epithelial cell marker E-caherin expression was significantly reduced.3.Irradiation may waken the proliferataion ability of the MCF-10 A but strengthen the move ability of the MCF-10 A and this effect could be blocked by phosphorylation inhibitor of the EGFR and Akt.4.After irradiation,the cell cycle was obviously blocked in G1 phase,the proportion of G1 phase was increased,which may indicates that the MCF-10A-10 Gy may reduce susceptibility to X-Ray.5.The application of the EGFR target drug may do benefit to patients with breast cancer.