The Effect Of Gefitinib On The Biological Behavior Of Breast Cancer Cells

EGFR is one of members of epidermal growth factor receptor(EGFR)family.It was the first discovered cellular surface signal transduction protein and pro-oncogene product,with the classic receptor features.EGFR is over-expressed in 14%～91%of breast cancer,and is highly relevant with poor prognosis.For this reason,the research and targeting therapy on EGFR pathway is promising in clinic.Gefitinib(Iressa)is one of small molecular tyrosine kinase inhibitors,which could competively compond with ATP-binding site to inhibit the autophosphorylation of EGFR,and block the down stream signal.Gefitinib has been wildly used in the NSCLC therapy at present,and the application on breast cancer is under basic and clinical research.In this thesis,we investigated the effects of gefitinib on the breast cancer cell biological behavior,including proliferation,apoptosis,movement and drug resistance,and provided the theoretical basis for clinical application.The EGFR expression positive cell lines,MDA-MB-231 and T47D,were chosen as cell models considering the western blotting result.The best EGFR activation concentration on 37℃ of EGF is 100ng/ml,and the stimulating time is 10min.Phosphorylation western blotting was used to identify the efficacy of gefitinib on EGFR pathway.The results demonstrated that gefitinib could targeting inhibit the EGFR phosphorylation levels of T47D and MDA-MB-231,and inhibit the most important down stream pathways,Ras/Raf/ERK and PI3K/Akt.But the activation of Akt and ERK1/2 could not be completely blocked by gefitinib,espescally on MDA-MB-231 cell line.MTT assays were used to evaluate the effect of gefitinib on the survival of MDA-MB-231 and T47D.At the concentrations of 0.1μM,1μM,10μM and 100μM,the inhibition rates on T47D cell line of gefitinib were(34.9±8.8)%,(42.6±5.0)%,(60.7±5.4)%and(89.2±0.2)%respectively,and the IC50 of gefitinib was 3.61 μM.the inhibition rates on MDA-MB-231 cell line of gefitinib were(24.8±4.9)%,(22.4±3.7)%,(48.3±5.9)%and(56.91±3.8)%respectively,and MDA-MB-231 cell line showed drug resistance to gefitinib.To explain the mechanism of inhibition,we tested the cell cycle treated with gefitinib using flow cytometry.Results showed the G0/G1 phase arrest of T47D and the restrict point cound not be surpassed.The apoptosis appearance of T47D cell line,treated with gefitinib,was observed under the microscopy.To verify the apoptosis,T47D treated with gefitinib was detected through flow cytometry(Annexin Ⅴ/PI).The apoptosis rates were 2.8%,72.8%and 94.9%respectively after Oh,12h and 24h incubation with gefitinib(10μM).Western blotting was used to investigate the molecular mechanism.As the increase of gefitinib concentration,the expression of Bcl-Xl decreased,and the expression of Bax increased.The cleavage of Caspase-3 and PARP also increased in the dose-depending method.Therefore,gefitinib could induce the T47D cell line apoptosis through the classic mitochondrial pathway.The effect of gefitinib on the cell movement(migration)was tested preliminarily by wound-healing assay and Boyden chamber assay.Comparing with control group,gefitinib could effectively enlong the wound-healing time and decrease the migration cell count.The effect displayed obvious dose-effect relationship.F-actin was further observed by immuno-fluoresence technique.Gefitinib could significantly inhibit the lamellipodium formation,F-actin polymerization and its polarity change.It may be the direct reason for the cell migration ability reduction.To study the effect on durg resistance of breast cancer cell,MTT assays and western blotting were applied to test the effects of gefitinib combined with Doxorubicin on MDA-MB-231 cell line.As a result,the IC50 and IC80 of Doxorubicin dropped as the combination of the two agents,and the cleavage of PARP also increased as the participation of gefitinib.Gefitinib could significantly enhance the cytotoxic effect of Doxorubicin,thus sensitize chemotherapy.In addition,the effect of Doxorubicin followed by gefitinib is much more powerful than simultaneous combination.On the contrary,the effect of gefitinib followed by Doxorubicin is less powerful than simultaneous combination.In conclusion,through the inhibition of the EGFR pathway activation,gefitinib could reduce the proliferation of breast cancer cells,induce apoptosis,decrease the ability of cell migration and sensitize chemotherapy.We believe that,gefitinib could provide a new method and idea for breast cancer therapy.But our experiments also demonstrated that,the downstreams of EGFR could not be blocked completely by gefitinib alone,and some of breast cancer cells may show a certain level of drug resistance.This may suggest the exsistance of drug resistance for gefitinib in clinic,and the necessary of the combination with other targeting drugs or chemotherapy.The different sequences of gefitinib and chemotherapy also induce different results.This highlights the choosen of drug administration sequence in clinic.