Study On The Effects And Mechanisms Of Eupatorium Lindleyanum DC’s Active Fraction Inhibiting The Prolifearation Of Triple Negative Breast Cancer(TNBC)Cells

Breast cancer is one of the most common malignant tumors of women,especially triple negative breast cancer(TNBC;lacking the expression of the estrogen receptor,progesterone receptor and human epidermal growth factor receptor 2)is a special subtype of breast cancer,because of the lack of specific targeted therapy,the poor prognosis and drug resistance.Therefore,the development of new chemotherapeutic agents has become very significant research for the prevention and treatment of TNBC.Eupatorium lindleyanum DC.(C ompositae),called ’Ye-Ma-Zhui’ by local residents,is a perennial herbaceous plant.It has antimicrobial,antihistamine and anti-inflammatory activities and is mainly used in the clinical treatment of respiratory diseases and hypertension.Literature research found that Eupatorium plants have anti-tumor activity.Therefore,it is hypothesized that Eupatorium lindleyanum DC.owns some chemical constituents with antitumor activity.This is the first study to investigate ethanol extract obtained from Eupatorium lindleyanum DC.on the proliferation of TNBC cells.We screened out the active fraction F1012-2 could significantly inhibit the proliferation of TNBC cells,the mechanisms of anti-proliferation were investigated in order to provide experimental basis and theoretical foundation for developing new anti-TNBC agents.Objective:The aim of this study was to investigate the growth inhibitory effect of F1012-2 on human triple negative breast cancer cells,and to elucidate its mechanisms through three aspects of cell cycle,apoptosis and autophagy.Methods:(1)We detected the effects of active fraction F1012-2 on the proliferation of TNBC(MDA-MB-468,MDA-MB-231)cells and human normal mammary cells MCF 10A by MTT assay.(2)We examined the effects of F1012-2 on cell cycle,apoptosis and mitochondrial membrane potential by flow cytometry(FCM).(3)We observed the effects of F1012-2 on autophagy by fluorescence microscopy.(4)We examined the expression of related proteins of cell cycle,apoptosis and autophagy by western blotting.(5)We first added the relevant inhibitors(Z-VAD-FMK,3-Ma),and then use flow cytometry to detect apoptosis and western blotting to detect the changes of related proteins.Results:(1)The active fraction F1012-2 can significantly inhibit the proliferation of TNBC cells in a time-and dose-dependent manner,but has no obvious toxic effect on human normal mammary cells MCF 10A.(2)The result of flow cytometry showed F1012-2 induces cell cycle arrest at G2/M phase,and the expression of cell cycle associated proteins cyclin B1,cdc2 and mutant p53 are downregulated,while p21,p27,p-cdc2(Tyr15)are upregulated.(3)We observe F1012-2 can activate caspase-9、caspase-8、caspase-3 and PARP,and it can induce caspase-dependent apoptosis as evidenced by the reversal effects of pan-caspase inhibitor Z-VAD-FMK on induction of apoptosis,the expression of cleaved caspase-3 and the cleavage of PARP.(4)F1012-2 can induce apoptosis with the loss of mitochondrial membrane potential.It also changes the expression of Bcl-2 family proteins with downregulating the expression of anti-apoptotic protein Bcl-xl and Bcl-2,while upregulating the expression of pro-apoptotic protein Bad and Bax.(5)Acridine orange(AO)staining shows that the degree of autophagy increased in a dose-dependent,with the accumulation of autophagy related protein LC3-Ⅱ and Beclin-1,downregulating the expression of p62.After adding autophagy inhibitor 3-Ma,the expression of LC3-Ⅱ is downregulated.(6)The rate of apoptosis is increased and the expression of cleaved caspase-3 and the cleavage of PARP are upregulated after adding autophagy inhibitor 3-Ma.(7)F1012-2 may regulate apoptosis by activating p38 and inhibiting Akt signaling pathway.Conclusion:The active fraction F1012-2 obtained from Eupatorium lindleyanum DC.can inhibit the proliferation of TNBC cells.The inhibitory effect may be regulated by cell cycle arrest,apoptosis and autophagy,furthermore,p38 and Akt signaling pathways are also involved in the process of apoptosis.Therefore,the active fraction F1012-2 inhibits the proliferation of TNBC cells through multiple signaling pathways,which is expected to become a potential agent for the treatment of TNBC.