Ailanthone Inhibiting Human Promyelocytic Leukemia HL-60 Cell Proliferation And Its Mechanism

Objective:The ailanthone is a natural small molecular compound of quassin extracted from the traditional Chinese medicinal Ailanthus altissima,has been thoroughly proven to have anti-tumor,anti-AIDS,anti-malarial,anti-allergic,anti-inflammatory and other activities.However,the anti-proliferative effect of ailanthone on human promyelocytic leukemia HL-60 cell line and its potential mechanism have not been reported.In this study,we conducted experiments by means of cellular molecular biology in order to discuss the inhibitory effect of ailanthone on proliferation of HL-60 cell line in vitro and its mechanism.Methods: The cell viability of HL-60 cell line was determined by MTT assay in vitro.Cells were stained with Annexin V-APC/7-ADD and the proportion of apoptosis was determined by flow cytometry.Cells were stained with Propidium iodide(PI)and Cell cycle changes were analyzed by flow cytometry.Cells were stained with acridine orange(AO)and were observed under electron microscopy to determine whether ailanthone could induce autophagy in HL-60 cells.The expression level of autophagy-related proteins was detected by Western blotting(WB).The role of ailanthone-induced autophagy in its inhibition of HL-60 cell proliferation was discussed by the addition of autophagy inhibitors.Results:The MTT assay showed that ailanthone was highly cytotoxic to HL-60 cells and inhibited cell proliferation in a dose-time dependent manner,the half-inhibitory concentrations(IC50)at 24,48,and 72 hours were 12.18,8.497,and 5.986 μmol/L,respectively.Annexin V-APC/7-ADD staining showed that ailanthone can induce apoptosis in HL-60 cells,and the number of apoptotic cells was in a dose-time dependent manner,After 5,10,20μmol/L ailanthone treatment with 48 hours,the apoptosis rate of each group was 42.02±0.54,52.05±2.27,59.69±0.25%;PI staining showed that ailanthone can cause cell cycle arrest in HL-60 cells by up-regulating the percentage of G0/G1 phasecells,The proportion of G0/G1 phase cells in each group was 53.54±0.88,58.42±0.31 and65.57±3.16%,respectively.AO staining suggested that ailanthone can induce the formation of acidic autophagic vesicles in the cytoplasm of HL-60 cells,which point that ailanthone can induce autophagy in HL-60 cells,and the pretreatment with BaF-A1 can significantly attenuate this process.WB showed that ailanthone up-regulated the protein expression levels of beclin-1 and LC3-II and down-regulated those of LC3-I and p62 in a dose-dependent manner.Use of BaF-A1 showed that the anti-proliferative effects of ailanthone on HL-60 cells may be partly attributable to the induction of autophagy-mediated apoptosis by MTT assay and annexin V-APC/7-ADD staining assay.Conclusion:Ailanthone has strong cytotoxicity to human promyelocytic leukemia HL-60 cells,which may be related to its induction of apoptosis,cycle arrest and autophagy in HL-60 cells.Ailanthone may induce autophagic cell apoptosis in HL-60 cells by promoting the expression of autophagy core protein beclin-1,inhibiting p62 protein expression,and promoting the conversion of LC3-I protein to LC3-II protein.