Investigation Of Apoptosis Induced By Combination Of Artesunate And Arsenic Trioxide Via Mitochondrial Apoptotic Pathway In NB4 Cells

ObjectiveIn this study,human acute promyelocytic leukemia cell line NB4 was used as the research object to investigate the effect of artesunate combined with arsenic trioxide on the apoptosis of NB4 cells.To explore whether the mitochondrial apoptotic pathway of human acute promyelocytic leukemia cell line NB4 was activated by the combination of artesunate and arsenic trioxide or not.the effects of artesunate and arsenic trioxide on the proliferation,cell cycle,apoptosis,mitochondrial membrane potential,mitochondrial ROS levels,related mRNA transcription levels and related proteins expression levels of NB4 cells were investigated.Methods1.The inhibitory effect of artesunate combined with arsenic trioxide on proliferation of NB4 cellsThe thiazolyl blue method was used to detect the effect of artesunate and arsenic trioxide alone or in combination on the proliferation of NB4 cells and the half inhibitory concentration was calculated.2.Effect of artesunate combined with arsenic trioxide on cell cycle and apoptosis of NB4 cellsFlow cytometry was used to detect the effect of artesunate and arsenic trioxide alone or in combination on cell cycle and apoptosis of NB4 cells.3.Effect of artesunate combined with arsenic trioxide on mitochondrial membrane potential of NB4 cellsThe JC-1 method was used to detect the effect of artesunate and arsenic trioxide alone or in combination on mitochondrial membrane potential of NB4 cells.4.Effect of artesunate combined with arsenic trioxide on mitochondrial reactive oxygen species in NB4 cellsThe MitoSOX method was used to detect the effect of artesunate and arsenic trioxide alone or in combination on mitochondrial ROS levels in NB4 cells.5.Effect of artesunate combined with arsenic trioxide on related mRNA transcription levels of NB4 cells qRT-PCR was used to detect the effects of artesunate and arsenic trioxide on the mRNA transcription levels of Bax and Bc1-2 in NB4 cells.6.Effect of artesunate combined with arsenic trioxide on related proteins expression levels of NB4 cellsThe effects of artesunate and arsenic trioxide on the expression levels of Bax,Bcl-2,Caspase-3,Caspase-9 and Cyt-C protein in NB4 cells were detected by Western blot.Results1.The inhibitory effect of artesunate combined with arsenic trioxide on the proliferation of NB4 cellsCompared with the control group,artesunate and arsenic trioxide showed significant value-inhibiting effect with concentration-dependent(P<0.05)on NB4 cells,respectively.The effect of combination therapy was much better than that of the single drug alone(P<0.05).2.The combination of artesunate and arsenic trioxide affects cell cycle and apoptosis of NB4After administration with artesunate and arsenic trioxide in NB4 cells,the proportion of G0-G1 phase was significantly higher than that of the control group(P<0.05),and the proportion of apoptosis was significantly higher than that of the control group(P<0.05).3.The combination of artesunate and arsenic trioxide affects mitochondrial membrane potential in NB4 cellsAfter administration with artesunate and arsenic trioxide in NB4 cells,the expression of mitochondrial membrane potential was significantly decreased compared with the control group(P<0.05).4.Effect of artesunate combined with arsenic trioxide on mitochondrial reactive oxygen species in NB4 cellsAfter administration with artesunate and arsenic trioxide in NB4 cells,the level of reactive oxygen species in mitochondria was significantly increased compared with the control group(P<0.05).5.Artesunate combined with arsenic trioxide affects related mRNA transcription levels in NB4 cellsAfter administration with artesunate and arsenic trioxide in NB4 cells,the transcription of Bcl-2 mRNA in NB4 cells was significantly decreased compared with the control group(P<0.05);while,the mRNA transcription of Bax was significantly increased(P<0.05).6.The combination of artesunate and arsenic trioxide affects the related protein expression levels in NB4 cellsAfter administration with artesunate and arsenic trioxide in NB4 cells,the expression level of Bcl-2 protein was down-regulated in NB4 cells compared with the control group(P<0.05);while,the expression levels of Bax,Caspase-3,Caspase-9 and Cyto-C were significantly increased(P<0.05).Conclusions1.Artesunate combined with arsenic trioxide has an inhibitory effect on NB4 cell proliferation and induces apoptosis.It has an anti-acute promyelocytic leukemia effect in vitro.2.Blocking the cell cycle in G0-G1 phase is one of the possible mechanisms of inhibition on cell proliferation by the combination of artesunate and arsenic trioxide.3.The apoptosis of NB4 cells induced by the combination of artesunate and arsenic trioxide may be activating the mitochondrial apoptosis signaling pathway.4.The increase of ROS concentration in mitochondria and the down-regulation of Bcl-2 protein expression and up-regulation of Bax protein may be the regulatory factors of mitochondrial apoptosis pathway induced by artesunate combined with arsenic trioxide in NB4 cells.