| Objective:To detect the main components of Sanhuang Decoction and construct a reasonable quality control system;establish a tamoxifen-resistant breast cancer cell line;and examine the effect of Sanhuang Decoction on the bio-behavior of tamoxifen-resistant cell lines.Explore the possible targets of Sanhuang Decoction.Method:1.Using high pressure liquid phase method to detect the components of three monomers of Sanhuang Decoction,detect the isoflavone glucoside at 260nm,detect curcumin at 430nm,and detect emodin at 254nm.2.CCK-8 assay was used to detect cell proliferation inhibition assay to detect the inhibitory effect of different concentrations of 4-OH tamoxifen on breast cancer MCF-7 and its inhibition on MCF-7 tamoxifen-resistant cells.The ability,the ratio of IC50 is the resistance index,and also used this method to detect the inhibitory effect of Sanhuang Decoction on tamoxifen-resistant cells.3.The apoptosis was observed by Hoechst 33258 staining,which visually showed the apoptosis of MCF-7 and MCF-7 tamoxifen-resistant cells under different concentrations of 4-OH tamoxifen.4.Using flow cytometry to detect the effect of different concentrations of different drugs on the cell cycle of different cells.5 Transwell assay was used to examine the effects of different drug interventions on the migration and invasion of tamoxifen-resistant cells.6.The effect of different drugs on the angiogenic ability of tamoxifen-resistant cells was examined using an experimental method of lumen formation.7.Using chicken embryo test,the effect of Sanhuang Decoction on angiogenesis ability of tamoxifen-resistant cells was detected in vivo.8.Western blot was used to detect PI3K signaling pathway and expression of migration,invasion and angiogenesis-related proteins.Results:1.We tested the three main components in Sanhuang Decoction by high pressure liquid phase method.In 500g Sanhuang Decoction,the content of emodin is 22±2.43μg·mL-1,and the content of curcumin is 25±1.67μg·mL-1,and the content of the isoflavone glucoside was 3 1 8±9.6μg·mL-1.In the subsequent basic experiments,we will use this as the quality control standard for Sanhuang Decoction.2.To inhibit the proliferation of breast cancer MCF-7 tamoxifen-resistant cells by different concentrations of 4-OH tamoxifen at 48h and the effect of 48h different concentrations of 4-OH tamoxifen on breast cancer MCF-7 cells.Proliferation inhibition showed that the IC50 value of 4-OH tamoxifen for MCF-7 cells was 2.48±1.06μM at 48 h,and the IC50 value for MCF-7/tamR cells was 32.73±2.19μM.According to the formula of the drug resistance index,the resistance index of MCF-7/tamR cells was 13.19±2.35 times.The detection of cell cycle further verified the successful construction of MCF-7 tamoxifen-resistant cells.The results of the experiment showed that MCF-7 cells were at GO in the presence of 4-OH tamoxifen at a concentration of 0-5μM for 48 h.The percentage of/G1 phase ranged from 52.42±4.21%to 83.49±1.62%,suggesting that 4-OH tamoxifen can induce MCF-7 cell arrest in GO/G1 phase,which is consistent with the literature.However,for the same concentration of 4-OH tamoxifen for 48h,there is no clear cycle block for MCF-7 tamoxifen-resistant cells,which can be considered at this concentration.4-OH tamoxifen has no cycle arrest effect on MCF-7 tamoxifen resistant cells.From this aspect,it can be further verified that the constructed MCF-7 tamoxifen resistant cells have certain resistance.At the same time,the results of Hoechst 33258 staining for apoptosis showed that for MCF-7 cells,as the concentration of 4-OH tamoxifen increased,the fluorescence intensity of the cells increased,and the nuclear cleavage and nuclear pyknosis were further advanced.Increase,showing that the degree of apoptosis increases with the increase of 4-OH tamoxifen concentration。but for MCF-7 tamoxifen-resistant cells,with the increase of 4-OH tamoxifen concentration,wither The degree of death has not changed much.Therefore,it can be determined by the above three experiments that tamoxifen-resistant cells of breast cancer have been successfully established.The expression of PI3K signaling pathway in MCF-7 tamoxifen-resistant cells was detected by Western Blot.The expression of PI3K,AKT and mTOR in MCF-7 tamoxifen-resistant cells was significantly higher than that in MCF-7 cells.Subsequently,with the use of agonists and inhibitors of the PI3K signaling pathway,it was further verified that the expression level of the PI3K pathway protein in MCF-7 cells after priming with the PI3K signaling pathway is essentially related to MCF-7 tamoxifen.The drug-resistant cells are consistent,and the expression of the PI3K signaling pathway-associated protein,which is highly expressed by MCF-7 tamoxifen-resistant cells,can also be inhibited by PI3K signaling pathway inhibitors.Therefore,we can conclude that one of the mechanisms of breast cancer MCF-7 tamoxifen resistance is excessive activation of the PI3K signaling pathxway.4.Sanhuang Decoction has a good inhibitory effect on breast cancer MCF-7 tamoxifen-resistant cells,and it is time-and concentration-dependent.The results of CCK-8 showed that the inhibition rate of Sanhuang Decoction on MCF-7 tamoxifen-resistant cells increased with increasing concentration within a certain time and range of action,and at the same drug concentration,Sanhuang Decoction the inhibitory rate of MCF-7 tamoxifen-resistant cells increased with time.Therefore,the inhibition rate of Sanhuang Decoction on breast cancer MCF-7 tamoxifen-resistant cells was positively correlated with time and concentration.At the same time,the percentage of MCF-7 tamoxifen-resistant cells in the S phase was from 38.32±2.14%to 56.71±3.62%after intervention of 0 mg/ml,4 mg/ml and 8 mg/ml of Sanhuang Decoction for 24 h.Sanhuang Decoction can induce breast cancer MCF-7 tamoxifen cell arrest in S phase,and its corresponding cyclin CDK2 and cyclinA,with the increasing concentration of tri-orange,protein expression has also been further reduced.5.Sanhuang Decoction has a certain inhibitory effect on the migration,invasion ability and angiogenesis ability of MCF-7 tamoxifen-resistant cells,and increases with the increase of concentration.6.Sanhuang Decoction reduced the expression of PI3K signaling pathway,migration,invasion and angiogenesis-related protein in MCF-7 tamoxifen-resistant cells,and the protein expression decreased further with the increase of the concentration of Sanhuang Decoction.7.The ability of Sanhuang Decoction to reduce migration,invasion and angiogenesis of MCF-7 tamoxifen-resistant cells is closely related to the ability of Sanhuang Decoction to reduce PI3K signaling pathway.The target of PI3K signaling pathway is PI3K signaling pathway.8.In the chicken embryo experiment,Sanhuang Decoction can inhibit the angiogenesis of chicken embryo chorioallantoic membrane caused by tamoxifen-resistant cells.Compared with the blank control group,the microvascular proliferation rate of the chicken chorioallantoic membrane in the tamoxifen-resistant cell group was 82.681±7.22%,and the tamoxifen given Sanhuang Decoction compared with the blank control group.The microvascular proliferation rate of chicken embryo chorioallantoic membrane in the drug-resistant cell group was 50.56±6.48%(P<0.05)Conclusion:Compared with MCF-7 cells,MCF-7 tamoxifen-resistant cells do have overexpression of the PI3K signaling pathway,which is consistent with the literature.Sanhuang Decoction can inhibit the proliferation,migration,invasion and angiogenesis of MCF-7 tamoxifen-resistant cells,and its clear target is the PI3K signaling pathway. |
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