Effect Of Z-Guggulsterone On Proliferation And Autophagy Of Breast Cancer MDA-MB-231 Cells And Its Molecular Mechanism

Background:Breast cancer is a common cancer threatening women’s health,and its incidence rate is increasing year by year.At present,the comprehensive treatment of breast cancer has made great success and progress,but there are still some breast cancers that are not sensitive to the treatment;Triple negative breast cancer(TNBC)has high malignant degree,strong heterogeneity,and is lack of HR receptor and effective target,so it can only be treated by cytotoxic drugs,which is a kind of breast cancer subtype with poor prognosis;Therefore,it is always one of the research focuses of breast cancer to find drugs with better effect and less side effects.In recent years,more and more attention has been paid to the relationship between autophagy and tumor.Autophagy plays an important role in the occurrence,development and treatment of breast cancer.Z-Guggulsterone is the main bioactive component of guggul,an Indian herbal medicine.Our previous studies found that Z-Guggulsterone has a certain inhibitory effect on breast cancer cells,but its exact efficacy and mechanism of action are not clear.In this study,autophagy will be used as a starting point to study the effect and mechanism of Z-Guggulsterone on breast cancer cell line.Obj ective:To explore the effect of Z-GS on the proliferation and autophagy of MDA-MB-231 cells,and explore its potential mechanism,so as to provide theoretical basis for the application of Z-GS in the clinical treatment of triple negative breast cancer.Methods:1.Effect of Z-GS on the growth activity of MDA-MB-231 cells:MTT Assay was used to detect the effect of different concentration of Z-GS(0,5,10,20,40 μmol/L)on the growth activity of MDA-MB-231 cells.The inhibition rates were calculated respectively,and the concentration inhibition rate curve was made according to the results.The concentration of half inhibition rate IC50 was calculated.2.Z-GS and the autophagy of MDA-MB-231 cells:The number of autophagosome in the cytoplasm of MDA-MB-231 cells treated with different concentrations of Z-GS(0,5,10,20,40μmol/L)for 24 hours was observed under fluorescence microscope;Western Blot was used to detect the expression of autophagy related protein LC3-Ⅱ.3.The mechanism of the autophagy of MDA-MB-231 cells affected by Z-GS:Western blot was used to detect the expression of AMPK-mTOR signal pathway related proteins p-AMPK and p-mTOR in MDA-MB-231 cells after 24 hours of treatment with different concentrations of Z-GS(0,5,10,20,40μmol/L);Using AMPK inhibitor compound C and Z-GS to treat MDA-MB-231 cells together,the changes of autophagosome were observed under fluorescence microscope and the expression of p-AMPK was detected by Western Blot.Results:1.Z-GS inhibits the proliferation of MDA-MB-231 cells.MTT results showed that Z-GS inhibited the growth activity of MDA-MB-231 cells in a concentration dependent manner.When MDA-MB-231 cells were treated by Z-GS for 24h,IC50(50%inhibition concentration)was 35.36μmol/L.2.Z-GS can induce autophagy of MDA-MB-231 cells.Under fluorescence microscope,we found that after Z-GS acted on MDA-MB-231 cells,the number of fluorescence spots increased in a concentration dependent manner,indicating that autophagosomes increased.Compare with MDA-MB-231 cells were treated only by Z-GS,When they were treated by both Z-GS and AMPK inhibitor compound C,the fluorescence spots decreased,indicating the decrease of autophagosomes.Western blot showed that the expression of LC3-Ⅱ increased in a concentration dependent manner when Z-GS treated MDA-MB-231 cells for 24 hours.These results suggest that Z-GS can induce autophagy of MDA-MB-231 cells,which can be reversed by AMPK inhibitor compound C.3.Z-GS may induce autophagy of MDA-MB-231 cells through AMPK-mTOR pathway.Western blot showed that when MDA-MB-231 cells were treated by Z-GS for 24 hours,the expression of p-AMPK increased and the expression of p-mTOR decreased in a concentration dependent manner;When they were treated by both Z-GS and Compound C,the expression of p-AMPK was slightly higher than that of Compound C group,and significantly lower than that of Z-GS group.Conclusions:1.Z-GS can inhibit the growth of MDA-MB-231 cells by inducing autophagy.2.Z-GS may induce autophagy in the cells through AMPK-mTOR pathway.