Inhibitory Effect Of MPD And VPA On Kasumi-1 Cells

Objective:The t（8;21）chromosomal translocation,associated with the acute myeloid leukemia（AML）subtype M₂ of the French–American-British classification,involves the AML1 on chromosome 21 and the ETO gene on chromosome 8.This translocation,one of the most frequent chromosomal anomaly in leukemia,generates an AML1-ETO fusion protein,which has the same runt homology domain（rhd）contained in AML1 protein.Thus,AML1-ETO that generates an AML1-ETO fusion protein is believed to act in part by repressing the transcription of AML1-driven genes associated with myeloid differentiation in hematopoiesis,inhibiting of hematopoietic stem/progenitor cells mature,which is one of the the precise mechanisms by which the fusion protein is involved in leukemia development.Therefore,the discovery of the drugs that can decrease AML1-ETO fusion protein is particularly important.To investigate the joint supression effect of glucocorticoids Methylprednisolone sodium succinate（MPD）and histone deacetylase（HDAC）inhibitor Valproic acid（VPA）in the treatment AML1-ETO postive t（8;21）/AML cell lines and its mechanism.Method:Kasumi-1 cell line–derived from t（8;21）AML was cultured,and treated with different concentration VPA,MPD for 3d,the IC₅₀（half maximal inhibitory concentration）of MPD and VPA were evaluated by MTT.Kasumi-1 cells were treated by control group,MPD group,VPA group and MPD+VPA group.RT-PCR and Western Blot were applied to detect the expression change of mRNA or protein level of AML1-ETO,AML1,ETO,GM-CSF,Bcl-2 and Bax.Result:1 MPD and VPA inhibited respectively the proliferation of the Kasumi-1cellThe Kasumi-1 cell growth was obviously inhibited with different concentration MPD or VPA after 3d in dose-dependent manner.The IC₅₀ of（half maximal inhibitory concentration）MPD and VPA on Kasumi-1 cell proliferation were（0.52±0.01）mmol/L and（2.31±0.01）mmol/L respectively.2 The influence of MPD,VPA,MPD+VPA group on expression of Bcl-2,Bax of Kasumi-1 cell2.1 The influence of MPD,VPA,MPD+VPA group on Bcl-2 mRNA,Bax mRNA of Kasumi-1 cellSemi-quantitation RT-PCR was applied to detect the expression of Bcl-2mRNA,Bax mRNA.Compared with control group,Bcl-2 mRNA expression was decreased and Bax mRNA expression was increased in 0.5mM MPD,3mM VPA and 0.5mM MPD+3mM VPA for 3d with statistical significance（P<0.01,vs control group）,MPD+VPA combined group was more effective than MPD,VPA single drug group（P<0.01,vs MPD+VPA group）,the same effective in MPD group and VPA group（P>0.05,MPD vs VPA group）.2.2 The influence of MPD,VPA,MPD+VPA group on Bcl-2 protein,Bax protein of Kasumi-1 cellWestern blot was used to detect the expression of Bcl-2 protein,Bax protein.Compared with control group,Bcl-2 protein expression was decreased and Bax protein expression was increased in MPD,VPA and MPD+VPA for3d respectively with statistical significance（P<0.01,vs control group）,and MPD+VPA combined group was more effective than MPD,VPA single drug group（P<0.01,vs MPD+VPA group）,the same effective in MPD group and VPA group（P>0.05,MPD vs VPA group）.3 The influence of MPD,VPA,MPD+VPA group on AML1-ETO protein,ETO protein of Kasumi-1 cellWestern blot was used to detect the expression of AML1-ETO protein,ETO protein.Compared with control group,AML1-ETO protein and ETO protein expression were depressed in MPD,VPA and MPD+VPA for 3d respectively with statistical significance（P<0.01,vs control group）,and MPD+VPA combined group was more effective than MPD,VPA single drug group（P<0.01,vs MPD+VPA group）,the same effective in MPD group and VPA group（P>0.05,MPD vs VPA group）.4 The influence of MPD,VPA,MPD+VPA group on expression of AML1,GM-CSF of Kasumi-1 cell4.1 The influence of MPD,VPA,MPD+VPA group on AML1 mRNA,GM-CSF mRNA of Kasumi-1 cellSemi-quantitation RT-PCR was applied to detect the expression of AML1mRNA,GM-CSF mRNA.Compared with control group,AML1 mRNA and GM-CSF mRNA expression were increased in MPD,VPA and MPD+VPA for3d respectively with statistical significance（P<0.01,vs control group）,and MPD+VPA combined group was more effective than MPD,VPA single drug group（P<0.01,vs MPD+VPA group）,the same effective in MPD group and VPA group（P>0.05,MPD vs VPA group）.4.2 The influence of MPD,VPA,MPD+VPA group on AML1 protein of Kasumi-1 cellWestern blot was used to detect the expression of AML1 protein.Compared with control group,AML1 protein expression was increased in MPD,VPA and MPD+VPA for 3d respectively with statistical significance（P<0.01,vs control group）,and MPD+VPA combined group was more effective than MPD,VPA single drug group（P<0.01,vs MPD+VPA group）,the same effective in MPD group and VPA group（P>0.05,MPD vs VPA group）.Conclusion:1.MPD and VPA inhibited respectively the proliferation of the Kasumi-1cell.2.The expression of proapoptotic gene Bcl-2 and antiapoptotic gene Bax were detected in Kasumi-1 cell respectively.The level of Bcl-2 mRNA and protein decreased after treatment with MPD and VPA,whereas increased Bax mRNA and protein.And MPD+VPA combined group was more effective than MPD,VPA single drug group by the change expression of Bcl-2 protein family,by which maybe induce the apoptosis mechanism of Kasumi-1 cell.3.MPD+VPA combined or single drug can decrease AML1-ETO fusion protein,and increase transcription of AML1 and up-regulate mRNA expression of AMLl target gene GM-CSF.AML1 protein expression was increased,but ETO decreased.In this series of experiments,we identified the combination treatment can significantly reverse the effects of AML1-ETO in t（8;21）/AML.Maybe ETO contained in AML1-ETO fusion dissociated.