Investigation Of Anticancer Effect Of Xanthoceraside In Vitro And The Mechanism Of Xanthoceraside-induced Human Breast Cancer Mcf-7 Cell Death

The present study was undertaken to investigate the anticancer effect of xanthoceraside in vitro, and thus its possible mechanisms involved in the potent antiproliferation effect on human breast cancer MCF-7 cell.The inhibition rate of different tumor cells and human peripheral blood lymphocyte cells was investigated by MTT assay.MCF-7 cells were stained with a combination of the fluorescent DNA-binding dyes acridine orange(AO)and ethidium bromide(EB)to investigate the morphology changes.Flow cytometry was employed to investigate the volume changes of MCF-7.The DNA agarose gel electrophoresis was further used to observe the DNA Fragmentation. Flow cytometry with propidium iodide-staining was consequently utilized to analyze the cell cycle distrubition of MCF-7 cells.Flow cytometry was employed to investigate the mitochondrial membrane potential with fluorescent cationic dye Rhodamine123.The antioxidant N-acetylcysteine (NAC)was then chosed to detect the influence on oxidant-stress system of MCF-7 cells. Necrostatin-1,a kind of cell necrosis inhibitor,was next chosed to detect the influence on antiproliferation effect of xanthoceraside-treated MCF-7 cells.As the results show,xanthoceraside could inhibit the prolifetation of tumor cells significantly in a dose-dependent manner in vitro,and the IC₅₀of different tumor cells were 5.10μM(HepG-2), 4.07μM(SGC-7901),3.64μM(A375-S2),4.56μM(A431)and 4.2μM(MCF-7).Under the concentration that the xanthoceraside could inhibit tumor cell significantly,there were not any cytotoxic effects on human peripheral blood lymphocyte cells.Cytoplasm vacuole and no significant condense of nuclear chromatin were observed under fluorescence microscopy after MCF-7 cells were exposed to xanthoceraside(9μM)for 0 hours to 12 hours,meanwhile,MCF-7 cells were bigger than the control group and smear was observed by agarose gel electrophoresis;The cell cycle distribution of MCF-7 was greatly changed after exposure to xanthoceraside(2.67-13.5μM)for 24h with an obvious G1 arrest compared with the control group.All that come to xanthoceraside induced MCF-7 cell necrosis.The mitochondrial membrane potential of MCF-7 showed significant decrease compared with the control group,and the antioxidant NAC attenuate the antiproliferation effect of xanthoceraside-treated MCF-7 cells and necrostatin-1(2.5-40μM)had no effects on the antiproliferation effect of xanthoceraside-treated MCF-7 cells,which might be the clue that the xanthoceraside-induced necrosis was dependent of mitochondria,meanwhile reactive oxygen species(ROS)participated in it.The xanthoceraside-induced MCF-7 cell death might not be the cell necrosis which initiated by Fas/TNFR and must be through RIP1 kinase.