Synergistic Inhibitory Effects On Acute Myeloid Leukemia U937 Cells By Combined Treatment Of HHT And As₂O₃

Objective:To investigate the effects and its related mechanism of the combination therapy with homoharringtonine?HHT? and arsenic trioxide?As₂O₃?on human myeloid cell line U937 in vitro researches.Methods:1 The proliferation of U937 cells was detected by MTT assay. 2 The cell apoptosis was determined by flow cytometry with Annexin-V-FITC/PI double staining.3 The protein expressions of p-AktSer473, p-AktThr308, Bcl-2, Bcl-XL, Bax, Bid,Mcl-1 and p-Mcl-1Thr163 in U937 cells were detected by Western blot.Results:1 Both HHT and As₂O₃ could significantly inhibit the growth of U937 cells in a dose-dependent manner. As the concentrations of HHT and As₂O₃ increased, the growth of U937 cells was inhibited more obviously. Moreover, U937 cells had a higher sensitivity to HHT. Compared with either drug alone, combination of HHT and As₂O₃ increased the inhibition rate in U937 cells.2 The combination of HHT and As₂O₃ could synergistically inhibited U937 cells.The proliferation inhibition rate of 30ng/ml HHT and 4?mol/L As₂O₃ were?51.81± 3.25?% and?17.03 ± 2.32?%, respectively. After treated with HHT combined with As₂O₃, the proliferation inhibition rate was?69.46±3.99?%.3 Both HHT and As₂O₃ could induce the apoptosis of U937 cells. The apoptosis rate of 30ng/ml HHT and 4?mol/L As₂O₃ were?5.37± 2.39?% and?26.47 ± 3.26?%for 24 h, respectively. The combined treatment of HHT and As₂O₃ increased the apoptosis rate to?35.42 ± 4.6?%.4 Western blot results showed that treated with either 4?mol/L As₂O₃ or 30ng/ml HHT for 8h, PI3K/Akt signaling pathways and the protein expression of Mcl-1 for16 h in U937 cells could be significantly inhibited. Treated with HHT or As₂O₃, theprotein expressions of PI3K/Akt signaling pathway and of Mcl-1 were down-regulated more and more obviously with time extending from 4 to 24 h.Treated with As₂O₃ for 4h, the protein expressions of p-Akt Ser473, Bcl-XL and p-Mcl-1Thr163 were significantly down-regulated. And the expression of p-AktThr308 was moderately down-regulated. After treated with 8h, the protein expression of Bcl-2 started to be down-regulated. And the expression of Mcl-1 was down-regulated only after 24 h. There were no significant changes both in Bid and in Bax expressions. The protein expressions of p-AktSer473, Bcl-XL and p-Mcl-1Thr163 were obviously down-regulated after treated with As₂O₃ for 4 to 16 h.The protein expression of p-AktSer473 was significantly down-regulated after treated with HHT for 8h. Compared with treatment of As₂O₃ alone, p-AktSer473 was reduced much later after treated with HHT. And the protein expressions of Mcl-1and Bcl-XL were significantly down-regulated after treated with HHT for 4h. The protein expressions of p-Akt Ser473, Mcl-1 and Bcl-XL were significantly down-regulated after treated with HHT for 4 to 16 h. Compared with treatment of As₂O₃ alone, the changes of the protein expressions of p-AktThr308, p-Mcl-1Thr163 and Bcl-2 were moderate after treated with HHT. There were yet no significant changes in Bid and Bax expressions after treated with HHT alone.Compared with treatment of HHT alone for 12 h, after the treatment of the both drugs, the expressions of Mcl-1, p-Mcl-1Thr163, Bcl-2 and Bcl-XL were down-regulated more significantly. And the changes of p-AktSer473 and p-AktThr308 after the combined treatment were as significant as HHT treated alone. In addition,neither the expression of Bid nor that of Bax changed markably after the combined treatment of the two drugs.Conclusion:The combination of HHT and As₂O₃ could synergistically inhibited U937 cells through inhibiting PI3 K / Akt signalingpathway and reduction of Mcl-1.