Effect of SARI Overexpression on Apoptosis of CBFL Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of SARI overexpression on the proliferation and apoptosis of core binding factor leukemia (CBFL) cells and explore the potential molecular mechanisms.@*METHODS@#C-KIT N822K mutation status in Kasumi-1 cell line was detected by exon 17 sequencing. Then the SARI lentiviral vector (pGC-FU-SARI) was constructed, meanwhile Kasumi-1 cells were transfected with the SARI lentiviral vector. Quantitative PCR and Western blot were employed to identify efficacy of SARI overexpression after the transfection of cells. Cells were divided into three groups, including the cells infected with pGC-FU-SARI (OE group), the cells infected with pGC-FU-GFP (NC group) and the untreated cells (blank control group). Cell proliferative activity was tested by MTT assay, cell apoptosis was measured by flow cytometry (FCM) and the expression of apoptosis-related proteins: BCL-2,BAX,Cyto C,Caspase 9,Caspase 3,cleaved-Caspase 3,PARP and cleaved-PARP as well as PI3K/Akt pathway proteins: PI3K（p85）,p-PI3K（p85）,Akt and p-Akt were detected by Western blot.@*RESULTS@#The Kasumi-1 cells were detected to bear c-KIT N822K (T>A) mutation. The Kasumi-1 cells with SARI was overexpression were construeted successfully. Compared with NC group, the cell proliferation was decreased and cell apoptosis was increased; BCL-2 expression was reduced, BAX expression was enharued; cyto C expression appeared; the expression of Caspase 9 and Caspase 3 was down-regulated, the expression of cleaved Caspase 3 was up-regulated; the PARP expression was decreased, cleaved PARP expression was increased; the phosphorylation level of PI3K/Akt pathway proteins: p-PI3K/PI3K, p-Akt/Akt was down-regulated in OE group (P<0.05).@*CONCLUSION@#SARI gene may suppress the proliferation of CBFL cells, and induce their apoptosis through the mitochondrial pathway, which may be related with the inhibition of PI3K/Akt pathway.

Effect of PI3K/Akt/mTOR signal pathway-mediated N822K mutation on apoptosis induced by c-KIT inhibitor in AML cells / 中国免疫学杂志

Objective:To investigate the effect of c-KIT N822K mutation on the apoptosis of AML cells induced by c-KIT inhibitor and to explore the underlying molecular mechanisms.Methods: Kasumi-1 cells that carry the c-KIT N822K mutation were used as experimental group,and HL-60 and NB4 cells with non-c-KIT N822K mutation were used as control group.These AML cells were treated with 0,0.04,0.16 and 0.64 μmol/L c-KIT inhibitor sunitinib for 24 h,respectively.Apoptosis-related proteins and PI3K/Akt/mTOR pathway proteins were detected by Western blot,compared the changes of cell-related signal pathway proteins in each group.Results: With the increase of sunitinib concentration,the expression of apoptosis-related proteins Bax,CytoC,Caspase-9, Actived-Caspase-3 and PARP in HL-60 and NB4 cells were increased (P<0.05),and the expression of anti-apoptotic protein Bcl-2 was down-regulated (P<0.01).However,the trend of this change was obviously weakened in Kasumi-1 cells with N822K mutation.In Kasumi-1 cells,the phosphorylation levels of PI3K/Akt/mTOR pathway proteins such as PI3K,Akt,4EBP1 and mTOR were down-regulated in a dose-dependent manner(P<0.05),but not in HL-60 cells and NB4 cells.Conclusion:The constitutive activation of c-KIT induced by N822K mutation may affect the apoptosis induction of c-KIT inhibitor sunitinib to Kasumi-1 cells,which may be related to the inhibition of PI3K/Akt/mTOR pathway.