The Growth Effects Of Diallyl Trisulfide On Acute Myeloid Leukemia Cells And The Mechanism

Objective To study the effects of diallyl trisulfide (DATS) on proliferation, apoptosis and cell cycle in acute myeloid leukemia cells HL-60 and the probable mechanism involved, aiming at offering credible academic and experimental bases for clinical use.Methods The HL-60 cells were co-incubated with DATS at various concentrations for different time. (1) Changes in the morphology of HL-60 cells were observed by light and fluorescence microscope. (2) The inhibition of cell growth was assessed by MTT spectrophotometric analysis. (3) The cell cycle distribution and apoptosis index of cells were measured by flow cytometry (FCM). (4) The telomerase activity was detected by TRAP-silver staining. (5) Immunohistochemical staining was used to detect the expressions of bcl-2, Survivin, COX-2 in cells. (6) The expression of cell cycle related protein was detected by western blotting.Results (1) In control group, the cells were big which the cell structure were complete, nucleolus and the membranes of nuclear were clear. But the HL-60 cells exhibited some morphologic features of apoptosis with 10mg/L, 20mg/L, 30mg/L DATS incubated for 24-48h , we can see the cells shrinkage, the proportion of nuclear and cytoplasm decreased, nucleolus was unclear in which chromatin concentrated to conglomeration assembled in the karyotheca or formed districted cresent and buddle in cytoplasm, apoptosis body. (2) DATS inhibited the growth rate of the cells obviously. The difference of inhibition rate between test group and control group was obvious (P<0.05). The inhibitory rate was increased significantly with the increase of concentration of DATS and the extension of time. (3) DATS changed the cell cycle distribution notably and caused a G2/M cell cycle arrest by FCM in a dose and time- dependent manner. The difference was significant (P<0.05). (4) Treated with DATS at 10-20mg/L for 24h and 48h can promote cells apoptosis of HL-60 cell significantly. (5) Treated with DATS(10mg/L,20mg/L,30mg/L) for 24-48h, the telomerase activity is inhibited obviously in a dose and time- dependent manner. (6) Compared with control group, the expression of bcl-2, Survivin, COX-2 decreased distinctly and the rate of expression decreased also in the test groups, the difference was significant (P< 0.05). (7) DATS could down regulate the expression of cyclinB1 protein but had little effect on the protein expression of cyclin-dependent kinase 1 (CDK1) in HL-60 cells by western blot.Conclusions (1) DATS can make acute myeloid leukemia cells HL-60 morphologic change. (2) DATS can inhibit growth and proliferation of the cells in a time and dose-dependent manner. (3) DATS changed the acute myeloid leukemia cells HL-60 cycle distribution notably, caused a G2/M cell cycle arrest in a manner of time-and-dose-dependency. (4) DATS may induce apoptosis of HL-60 cells in a manner of time- and concentration-dependency. (5) The mechanism of DATS inhibiting HL-60 growth, proliferation and inducing apoptosis is correlated with down regulation of bcl-2, Survivin and COX-2, DATS can inhibit the telomerase activiy of HL-60 cells. (6) DATS could down regulate the expression of cyclinB1 protein but had little effect on the protein expression of cyclin-dependent kinase 1 (CDK1) in HL-60 cells.