Combined Treatment Of Homoharringtonie And Arsenic Trioxide Has Synergistric Killing Effects On U937 Cells Co-Cultured With HS-5 Cells And Its Mechanism

Objective: To investigate the effect of Homoharringtonine?HHT? in combination with Arsenic trioxide?As₂O₃? on U937 cells co-cultured with HS-5cells and its related mechanisms.Methods: MTT method was used to detect the cell proliferation. The cell apoptosis was determined by flow cytometry with AnnexinV-PE/7AAD double staining. Mitochondrial membrane potential was measured by JC-1 staining using flow cytometry. The expression of p-AktSer473,p-Gsk-3?,Mcl-1, p-Mcl-1Thr163,Bcl-2, Bcl-xl,PARP, Caspase3 and Caspase9 were evaluated by Western blot.Results: As₂O₃ or HHT remarkably inhibited proliferation of U937 cells cultured alone respectively in time- and dose-dependent manner. As compared with the U937 cells cultured alone, the sensitivity of the U937 cells co-cultured with HS-5 to As₂O₃ or HHT was reduced. After treated with As₂O₃, the inhibition rate were 18.18% VS 26.50%?4?M?, 24.86% VS 40.74%?6?M? and 28.25% VS44.12%?8?M? respectively. While after treated with HHT,t he inhibition rate were4.59% VS 23.45%?10ng/ml?, 25.18% VS 55.67%?20ng/ml? and 59.95% VS78.82%?40ng/ml? respectively.Compared with treatment of either As₂O₃ or HHT alone, combined treatment of the two drug could exert more significant inhibitory effects on the survival of U937 cells co-cultured with HS-5. Results of the analysis by Jin's formula showed that the combined treatment demonstrates synergistic killing ability, Results also showed that the combination of 20ng/ml HHT and 8?M As₂O₃ exerted the most significant advantages of synergism. Therefore, the above combined treatment were used in the following experiments.HHT could induce apoptosis in co-cultured U937 cells. After treated withHHT for 8, 16 and 24 h, the apoptosis rates of co-cultured U937 cells were 13.75%,20.40% and 35.51%, respectively. After treated with As₂O₃ for 8, 16 and 24 h, the apoptosis rates of co-cultured U937 cells were 3.75%, 5.91% and 9.46%,respectively. Combined treatment with HHT and As₂O₃ could significantly increased the apoptosis rates, reached 22.35%, 27.98% and 44.82% after treated for 8, 16 and 24 h, respectively. After treated with HHT for 4 and 8h, the mitochondrial inner membrane potential? ? ?m? in the co-cultured U937 cells decreased significantly and the percentage of cells with depolarized mitochondrial increased to 11.68% and 18.44%, respectively. The ??m significantly decreased in the co-cultured U937 cells after treated with As₂O₃ for 8h?not for 4h?, and the proportion of cells with depolarized mitochondrial was 10.57%. After combined treatment with HHT and As₂O₃ for 4 or 8h, the ? ?m significantly reduced and the percentages of cells with reduced depolarized mitochondrial were16.91% and 25.68%, respectively.Compared with the U937 cells cultured alone, the protein expression levels of Mcl-1, Bcl-xl in co-cultured U937 cells were up-regulated. And there was no significant change in Bcl-2.The PI3K/Akt signaling pathway was activated with upregulated expression of p-AktSer473.When co-cultured U937 cells were treated with 20ng/ml HHT for 4 to 24 h,the activated PI3K/Akt signal pathway was inhibited with down-regulating the expression of p-AktSer473 and p-Gsk-3?. At the same time, mitochondrial apoptotic pathway was activated with the cleavage of PARP, Caspase3 and Caspase9.While there was no remakable change in p-Mcl-1Thr163 and Bcl-2.When co-cultured U937 cells were treated with As₂O₃ for 16 h, the activated PI3K/Akt signal pathway was inhibited with down-regulating expression of p-AktSer473 and there was cleavage in PARP. The activity of Caspase3 and Caspase9 were enhanced. There was no significant change in Mcl-1, Bcl-xl, p-Gsk-3?,p-Mcl-1Thr163 and Bcl-2.Compared with the treatment with either single drug, combined treatment with As₂O₃ and HHT could more significantly inhibit PI3K/Akt signal pathwayand activate mitochondrial apoptotic pathway, down-regulating the expression of Mcl-1, Bcl-xl and p-Mcl-1Thr163. And there were more cleavages in PARP and Caspase 9.Conclusion:The combination of As₂O₃ and HHT has synergistic killing effect on U937 cells co-cultured with HS-5 cells.