LY294002对CML急变期细胞中Wnt/β-catenin信号通路的影响及其效应

Effect of LY294002 on Wnt/β-catenin Pathway in CML Blastic Cells

β-catenin在慢性粒细胞白血病急变过程中发挥着重要作用,而其受BCR/ABL及其下游信号通路调控的具体分子机制尚未完全阐明。该研究旨在探讨PI3K-AKT信号通路对慢粒急变期细胞的影响及其对Wnt/β-catenin信号通路的调控作用。采用PI3K-AKT信号通路的靶向抑制剂LY294002作用于慢粒急变期K562细胞,MTT法检测其对细胞增殖的影响,甲基纤维素克隆形成实验检测细胞的克隆形成能力,Western blot检测pAKT(Thr308)的表达变化,RT-PCR和Western blot分别检测β-catenin及其下游靶基因c-myc、cyclinD1的mRNA和蛋白表达情况。结果显示,10,20,40μmol/L的LY294002作用细胞24 h后,抑制了K562细胞的增殖以及克隆形成能力,该效应呈浓度依赖的方式。3种浓度的LY294002处理细胞后,PI3K-AKT信号通路明显被抑制,pAKT(Thr308)的蛋白表达明显减少;β-catenin的mRNA表达无明显改变,但其蛋白水平依次减少;β-catenin的下游靶基因c-myc、cyclinD1的mRNA和蛋白水平均明显降低。综上所述,抑制PI3K-AKT信号通路可抑制白血病K562细胞的增殖和克隆形成能力,其机制可能与抑制Wnt/β-catenin信号通路相关。

β-catenin plays important roles in the progression of chronic myeloid leukemia blasts, whereas the relationship between β-catenin and the key pathways activated by BCR/ABL have not yet been elucidated. To investigate the impact of PI3K-AKT pathway on Wnt/β-catenin signal pathway, PI3K-AKT inhibitor LY294002 was used in CML blastic cells K562. Cell growth was detected by MTT test. The ability of cell colony was assessed by colony-forming assay. The protein expression of pAKT （Thr308） was detected by Western blot. The mRNA and protein expressions of β-catenin and its down-stream targets c-myc and cyclinD1 were analyzed by RT-PCR and Western blot, respectively. As a result, LY294002 significantly inhibited K562 cell growth and colony-forming ability in a dose-dependent manner. Meanwhile, the protein levels of pAKT （Thr308） and β-catenin were decreased in a dose-dependent manner after LY294002 treatment for 24 h, while the mRNA offl-catenin was not affected. Compared with the control groups, the mRNA and protein levels of c-myc and cyclinD1 were also decreased obviously after PI3K inhibitor treatment. Our data indicated that blockade of PI3K-AKT signaling inhibited the proliferation of CML blastic cells and the mechanism might be related to down-regulated expression of Wnt/ β-catenin pathway.