蛋白磷酸酶C1-TEN在急性髓系白血病细胞中的表达及抑制FLT3-ITD对其表达的影响

The expression of protein phosphatase C1-TEN in acute myeloid leukemia cells and inhibition of FLT3-ITD on its expression

目的:本研究旨在揭示急性髓系白血病(AML)细胞中具有C1结构域并与TENsin同源的蛋白磷酸酶(C1-TEN)的表达情况,并探讨中间串联重复突变FMS样酪氨酸激酶3(FLT3-ITD)抑制条件下C1-TEN的表达水平。方法:利用酪氨酸激酶抑制剂(TKI)索拉菲尼、米朵妥林作用于AML细胞系HL-60细胞、MV4-11细胞,采用MTT实验评价AML细胞对TKI的敏感性;TKI对AML细胞FLT3活化作用及对C1-TEN表达的影响采用Western blot(WB)分析;CCK-8实验分析C1-TEN抑制对AML细胞活性的影响;WB、流式(FACS)检测细胞凋亡情况。结果:相比FLT3野生型(FLT3-WT)HL-60细胞,FLT3-ITD突变MV4-11细胞对TKI显著敏感(P<0.05)。WB结果显示,经TKI作用后MV4-11细胞的酪氨酸激酶FLT3活化较HL-60细胞显著下降(P<0.05)。与阴性对照组比较,TKI处理组MV4-11细胞C1-TEN表达显著下降,而HL-60细胞的变化不明显。C1-TEN抑制剂DHTS作用后,MV4-11细胞活性显著下降(P<0.05),且DHTS与TKI具有协同抑制作用。WB、FACS结果显示,DHTS处理组MV4-11细胞凋亡显著高于阴性对照,差异具有统计学意义(P<0.05)。结论:本研究首次报道AML细胞C1-TEN表达情况,揭示FLT3-ITD能上调AML细胞C1-TEN表达,而抑制C1-TEN可通过细胞凋亡机制杀伤FLT3-ITD突变的AML细胞,因此提示该蛋白磷酸酶可能是FLT3-ITD下游分子,有望成为AML治疗的新靶点。

Objective:The aim of this study was to reveal the expression of C1 domain-containing phosphatase and TENsin homologue(C1-TEN)in acute myeloid leukemia(AML)cells,and to investigate the expression level of C1-TEN under the inhibition of FMS-like tyrosine kinase 3 internal tandem duplication(FLT3-ITD).Methods:AML cell lines HL-60 cells and MV4-11 cells were treated with tyrosine kinase inhibitors(TKI)Sorafenib and middotropin,and the sensitivity of AML cells to TKI was evaluated by MTT assay.The effects of TKI on FLT3 activation and C1-TEN expression in AML cells were analyzed by Western blot(WB).The effect of C1-TEN inhibition on AML cell activity was analyzed by CCK-8 assay.WB and FACS were used to detect cell apoptosis.Results:Compared with FLT3 wild-type(FLT3-WT)HL-60 cells,FlT3-ITD mutant MV4-11 cells were significantly sensitive to TKI(P<0.05).The results of WB showed that the activation of tyrosine kinase FLT3 in MV4-11 cells after TKI treatment was significantly decreased compared with that in HL-60 cells(P<0.05).Compared with the negative control group,the expression of C1-TEN in MV4-11 cells decreased significantly in TKI treatment group,while the change in HL-60 cells was not obvious.After the treatment of C1-TEN inhibitor DHTS,the activity of MV4-11 cells was significantly decreased(P<0.05),and DHTS and TKI had synergistic inhibitory effect.The results of WB and FACS showed that the apoptosis of MV4-11 cells in DHTS treatment group was significantly higher than that in negative control group,the difference was statistically significant(P<0.05).Conclusion:The expression of C1-TEN in AML cells was reported by this study for the first time,revealing that FLT3-ITD can up-regulate the expression of C1-TEN in AML cells,while inhibition of C1-TEN can kill FLT3-ITD mutant AML cells through apoptosis mechanism.It was suggested that this protein phosphatase may be the downstream molecule of FLT3-ITD,expecting to be a new target for the treatment of AML.