Effects Of Saikosaponin D On Apoptosis And Autophagy In Triple-negative Breast Cancer Cells MDA-MB-231

Breast cancer remains one of the most common cancers threatening women's health worldwide.Its morbidity and mortality are expected to continue to increase in the coming years.Triple-negative breast cancer accounts for less than a quarter of those cancers.Tri-negative breast cancer is more aggressive than other subtypes of breast cancer,may spread through local infiltration or cause distant metastasis through blood and lymph circulation,and is characterized by high recurrence rates.Autophagy is involved in a variety of physiological functions and is considered to be a key process for maintaining homeostasis in cells.At the same time,autophagy is also an effective cancer escape mechanism,which is associated with drug resistance of many cancers.Autophagy is usually initiated by the synergistic action of several autophagy-related proteins.Part of cytoplasm and organelles are wrapped in the double-membrane autophagosome.A variety of studies have shown that TCM treatment has the advantages of reducing the adverse reactions and drug resistance during traditional treatment to a certain extent.Bupleurum saponin D(SSD)is a triterpenoid saponin extracted from Bupleurum.It has been reported that the function of Saponin D is very complex,and it has been found to inhibit the proliferation of a variety of tumor cells,including ovarian cancer,lung cancer,liver cancer and glioblastoma.Purpose:The purpose of this study was to investigate the effects of Saikosaponin D(SSD)on apoptosis and autophagy of MDA-MB-231 in human breast cancer.Material and method : Cells were cultured in 96-well plates with different concentrations of drugs for 12,24,and 48 h,respectively.Then,the cells in each well were treated with CCK-8 solution,incubated at 37? for 2 hours,and the absorbance value was measured at 450 nm using a microplate analyzer.When the cell growth reached about 70%,it could be treated with drugs for 24 hours,and then stained with FITC-Annexinv and propidium iodide,and then analyzed by Fflowjo software.The autophagosomes were observed under electron microscope.Proteins were separated by SDS-PAGE gels at different concentrations.They were fixed with 4% oligoformaldehyde before photographing.Finally,the images were captured by fluorescence microscope.Results:1.After SSD treatment,the IC50 value was 5.49?M at 12 h.The IC50 value at 24 h was 6.85?m.The IC50 value at 48 hours was 10.41 ?m.2.Flow cytometry showed that the total percentage of early and late apoptosis in the high concentration group(8 ?m)was significantly higher than that in the control group(P<0.05),there was no significant difference between low concentration group(4?M)and medium concentration group(6?M)treatment groups compared with control group(P>0.05)3.SSD induced a large number of intracellular vacuoles in MDA-MB-231 cells within 12 h.4.The number of yellow and red dots in RAPA-treated cells(positive control)was increased by confocal laser microscopy.In the case of SSD treatment,we observed the formation of a large number of red and green dots in the cells,with a yellow overlay.5.LC3B ? expression as the drug concentration and time increased,reached a peak in 12 h after falling for positive control(RAPA).The expression of P62 increased with the increase of drug concentration,while Atg7 and Beclin1 did not change.The expression of LC3B in cells treated with SSD and CQ was higher than that treated with SSD and CQ alone.Conclusion:Our data suggest that SSD can induce apoptosis of MDA-MB-231 cells and inhibit the formation of autophagosomes by inhibiting the fusion of autophagosomes and lysosomes.This provides further evidence for the association between inhibition of autophagy degradation and cell death.