| Objective:In this study,we mainly focus on the investigation of Babao Dan(BBD)agent on its inhibiting effects to the proliferation of the mouse breast cancer 4T1 cells via the AKT and ERK signaling pathways,the project aims to provide experimental evidence for the prevention and treatment of breast cancer in clinics.Methods:1.The mouse breast cancer 4T1 cells viability was conducted with CCK-8 assay to study the effects of BBD on 4T1 cells after different concentrations(0,0.25,0.5 and 0.75 mg/m L)and different time(24 h,48 h,72 h)incubation.2.The mouse breast cancer 4T1 cells were cultured.The cell growth and confluence were observed under an inverted microscope,and the cell number was detected by trypan blue staining after different concentrations of BBD(0,0.25,0.5 and 0.75 mg/m L)interventions.The effect of BBD intervention on the growth and number of 4T1 cells after 24 h,48 h and 72 h were observed.3.After 24 hours of serum-free culture of the mouse breast cancer 4T1 cells,fetal bovine serum was used to intervene the cells at different time points(0,5,15,30,60 and 120 min),and Western blot was used to detect the phosphorylation of AKT and ERK proteins.Furthermore,the mouse breast cancer 4T1 cells were cultured and divided into control group,model group,0.5 mg/m L BBD group,0.75 mg/m L BBD group,U0126 group(ERK pathway inhibitor)and LY294002 group(AKT pathway inhibitor).Western blot was used to detect the phosphorylation of AKT and ERK proteins after the BBD intervention.4.The mouse breast cancer 4T1 cells were cultured and divided into 8 groups: Control group,BBD group,U0126 group,LY294002 group,BBD+U0126 group,BBD+LY294002group,U0126+LY294002 group and BBD+U0126+LY294002 group.After the intervention,the cell number change in each group were detected by trypan blue staining and observed under an inverted microscope independently.Results:1.The CCK-8 assay result preliminarily suggested that different concentrations of BBD could inhibit the proliferation of the mouse breast cancer 4T1 cells in different time points.Compared with the control groups,after treatment with different concentrations of BBD(0,0.25,0.5 and 0.75 mg/m L)for 24 h,48 h and 72 h,the cell viability of each time points was significantly decreased with the increase of BBD concentration,which was in a dose-dependent manner.The difference was statistically significant(P<0.001).2.The morphology of the mouse breast cancer 4T1 cells were observed through the inverted microscopes.In the control group,it showed that the mouse breast cancer 4T1 cells closely arranged,full in shape,clear in boundary,clumped into sheets,and fusiform in distribution.The nucleus and cytoplasm are clear,and the refraction is good.On the contrary,with the increase of BBD concentration,the cells in each group grew slowly and dispersed,and the intercellular space widened,and the growth state was significantly inhibited.Compared with the control group,the results of trypan blue staining showed that the cell number of each group decreased significantly in a dose-dependent manner with the increase of BBD concentration,and the difference was statistically significant(P<0.001).The results at different time points(24 h,48 h and 72 h)were consistent with the above conclusions,which further suggested that different concentrations of BBD could inhibit the proliferation of4T1 cells in different time points.3.Western blot results showed that the phosphorylation of ERK and AKT protein in mouse breast cancer 4T1 cells was significantly upregulated after fetal bovine serum intervention,the expression levels of both proteins reached the peak value at 5 min and 30 min.Therefore,5 min and 30 min were used as the optimal time for subsequent activation of ERK and AKT signaling pathways,respectively.Additionally,in the BBD intervention experiment,compared with the control group,different doses of BBD could significantly inhibit the phosphorylation of ERK and AKT proteins,which were equivalent to those in the U0126 and LY294002 groups.These results suggest that FBS can activate the phosphorylation of ERK and AKT proteins,and different concentrations of BBD can dose-dependently inhibit the phosphorylation of ERK and AKT proteins that activated by FBS.4.Observation under an inverted microscope showed that compared with the control group,the growth rate of the mouse breast cancer 4T1 cells in different groups slowed down,and the intercellular space widened,especially in the U0126+LY294002 group and the BBD+U0126+LY294002 group.The trypan blue staining experiment showed that compared with the control group,the cell number of mouse the breast cancer 4T1 cells decreased significantly after intervention of 0.5 mg/m L BBD for 24 h,48 h and 72 h,the difference was statistically significant(P<0.001).In particular,there was no significant difference in the inhibition of proliferation between the BBD+U0126+LY294002 group and the U0126+LY294002 group,which was not statistically significant(P>0.05).In summary,the inhibition of BBD on the proliferation of the 4T1 cells was achieved through ERK and AKT signaling pathways.Conclusion:BBD can reduce the viability of the mouse breast cancer 4T1 cells,inhibit cell growth,and reduce the cell number,thereby inhibiting the proliferation of the mouse breast cancer4T1 cells,and BBD inhibits the phosphorylation of AKT and ERK proteins that induced by FBS.In summary,BBD inhibits the proliferation of the mouse breast cancer 4T1 cells through AKT and ERK signaling pathways. |
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