| Object:To research the antitumor activity and its relevant medicine mechanisms of capecitabine(Cap)combined with fulvestrant(Ful)was used to human breast cancer MCF-7 cells.Methods:1.To discover the cell viability of human breast cancer MCF-7 cells treated by the different concentrations of Cap with using the method of CCK-8,and received IC50;2.Set up a blank control group,ful monotherapy group,cap monotherapy group and Cap+ful united group,the drug effect after 48 hours of detection on the proliferation of MCF-7 cell line inhibition,and according to OD value calculation cell growth inhibition rate and the two drugs combined q value;3.By Colony formation assay to detect the sensitivity of MCF-7 cell to drugs;4.Using the Hochest33342 fluorescent staining method to dye MCF-7 cells detecting the status of morphological changes of apoptotic cell nucleus;5.By flow cytometry assay to probe cell apoptosis and cell cycle distribution situation of MCF-7 cell made by different concentrations of Cap and Ful;6.Using the different concentrations of Cap and Ful to explore the expression of relevant apoptosis and cycle protein of breast cancer MCF-7 cell by Western blot detection mode,such as P53、pro-caspase3、PARP、cyclinB1 and cdc2.Results:1.For capecitabine to MCF-7 cell growth inhibition with culture medium diluted in a series of different concentrations for grow function in breast cancer cell MCF-7 after 24、48、72 hours have an impact on the cell proliferative inhibition and present a concentration and time dependency(P<0.01);2.After 48 hours,the inhibition rate of different concentrations of Cap(0.5、1、2、4、8μM)to MCF-7 cell were(25.71±0.49)%、(31.75±0.74)%、(40.94±0.5)%、(63.78±1.69)%、(89.58±1.02)%respectively.The IC50 of Cap is 2.714μM;3.After using Ful(5μM)、Cap(0.5μM)、Cap(0.5μM)and Ful(5 μM)、Cap(2μM)、Cap(2μM)and Ful(5μM)treated MCF-7 cell,the inhibition rate were(6.34±0.53)%、(26.45±0.29)%、(31.31 ± 1.31)%、(38.64±0.7)%、(43.68 ±0.73)%respectively;4.According to the result of Colony formation assay,we know that the MC F-7 cell to Cap was sensitive,When the concentration of Cap more than 0.5μM,the clone formation rate was very low;5.With the Hochest fluorescence staining inspection method,the experimental result presented that MCF-7 cell made by different concentrations of Cap and Ful appeared the typical nucleus morphology of apoptotic cell under the fluorescence microscope;6.After treated 48 hours,by the flow cytometry analysis,we discovered that Ful、Cap-L+Ful、Cap-H and Cap-H+Ful could induced apoptosis of MCF-7 cell,and the apoptosis rate is(6.23±1.49)%、(8.13±0.15)%、(9.73±1.20)%、(14.8±0.89)%、(12.17±1.89)%、(24.57±1.19)%(P<0.01).The rate of G1 and S phase are decrease,the rate of G2/M phase is increase.With the Western blot to detect apoptosis and cycle related protein of MCF-7 cell,the result show that the pro-caspase3 and PARP were decreased,P53 and cleaved-PARP were increased,the cyclinB1 and cdc2 were increased,and compared with the monotherapy group,the combined group were more obviously.Sometimes the expression of ER was decreased in the combined group.Conclusion:Capecitabine could significantly inhibit the proliferation of MCF-7 cell,and had a concentration and time dependent.Fulvestrant could enhanced the inhibition that capeicitabine to MCF-7 cell.By the experiment we can detect that the mechanism of medicine effect may have something to do with the occurrence of cell apoptosis and retardarce of cell G2/M-phasel cycle;The study result provided the experimental basis for the clinical application of capeicitabine combined with fulvestrant treated ER positive breast cancer. |
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