| Objective: To investigate whether homoharringtonine(HHT)could potentiate the anti-leukemia activity of arsenic trioxide(ATO)in HL-60 cells adhered to stomal HS-5 cells and the role of Mcl-1 in it.Methods: The inhibitory effect of ATO(4,8,16?M)or HHT(2,4,8ng/ml)alone on proliferation of HL60 cells before and after co-culture with HS-5 cells,8?M ATO and 8ng/ml HHT alone or combination on proliferation of co-cultured HL60 cells was detected by CCK-8 method and Colony formation assay.AnnexinV-PE/7AAD double staining and mitochondrial membrane potential flow cytometry were used to detect the apoptosis of co-cultured HL60 cells treated with 8?M ATO or 8ng/ml HHT alone or combination.The expression of t-Akt,p-AktSer473,Gsk-3?,p-Gsk-3?,Mcl-1,Bcl-2,PARP,Caspase3 and Caspase9 were evaluated by Western blot.RT-qPCR was used to detect the level of mRNA in mono-cultured and co-cultured HL60 cells and which treated with 8ng/ml HHT.The effect of 8ng/ml HHT on the degradation of Mcl-1 protein in co-cultured HL60 cells was studied by proteasome inhibitor experiments.The Mcl-1 gene in HL60 cells was silenced by RNAi,and the inhibitory effect of ATO(4,8,16?M)on proliferation of this cells was detected by CCK-8 method.1.ATO or HHT alone could significantly inhibited the proliferation of HL60 cells in a time-and dose-dependent manner.However,the sensitivity to ATO decreased significantly after co-cultureing.After treated with 4?8 and 16?M ATO respectively,the inhibition rate of mono-cultured and co-cultured HL60 cells were 9.20±1.83% vs-1.99±1.63%?18.30±2.91% vs 0.65±0.27% and 24.61±2.96% vs 5.93±4.13%.HHT potentiated the anti-leukemia effect of ATO on co-culture of HL60 cells.Co-cultured HL60 cells were treated with 8?M ATO or 8ng / ml HHT alone or combination for 24 h,the inhibition rate were 0.65±0.27% ? 28.11±5.48% and 46.23±4.36% respectively.The combination of HHT and ATO could significantly inhibit the colony formation capacity of co-cultured HL-60 cells.2.The combination of HHT and ATO could significantly decrease the mitochondrial membrane potential and induce the apoptosis of co-cultured HL-60 cells.Compared with ATO or HHT alone,the combination of the two drugs had a stronger activating effect on the mitochondrial apoptosis signal pathway in co-cultured HL60 cells with increased PARP apoptotic fragments and caspase 9 activation fragment,significant downregulation of p-Gsk-3? and Mcl-1.3.The combination of HHT and ATO could significantly inhibit primary AML cells.Treated with 2?M ATO or 60ng/ml HHT alone or in combination for 24 h,the inhibitory rates on primary AML cells were-1.57 ± 1.53%,31.27 ± 15.43% and 63.98 ± 18.99%,respectively.Compared with HHT or ATO alone,the combination of HHT and ATO could induce apoptosis and down-regulateMcl-1 expression in primary AML cells.4.Co-culture with bone marrow stromal HS-5 cells could up-regulate the expression of Mcl-1 protein in HL60 cells.The expression of Mcl-1 and Bcl-2 protein was significantly down-regulated in co-cultured HL60 cells treated with 8ng/ml HHT for 24 h.However,the level of Mcl-1 mRNA did not change in mono-culture and co-culture HL60 cells.Moreover,treated with 8ng/ml HHT for 24 h,the level of mcl-1 mRNA in co-culture HL60 cells do not change,which suggests that HHT down-regulates the expression of Mcl-1 in co-culture HL60 cells in post-translational or post-translational levels.5.HHT decreased the expression of Mcl-1 in HL60 cells through proteasome degradation pathway and inhibition of Mcl-1 phosphorylation at Thr163 site.6.The sensitivity to ATO in HL60 cells was significantly increased after RNAi silencing Mcl-1 in HL60 cells.Moreover,the sensitivity to ATO in HL60 cell do not change after co-culture with bone marrow stromal HS-5 cells.Conclusion: Co-culture with stroma cells reduced the sensitivity to ATO in HL60 cells.HHT potentiate the anti-leukemia activity of ATO through inhibiting Mcl-1. |
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