三氧化二砷联合普纳替尼对白血病细胞KG-1的抑制效应

Effects of the arsenic trioxide and ponatinib against human leukemic KG-1 cells

目的:探讨小分子酪氨酸激酶抑制剂普纳替尼联合三氧化二砷(arsenic trioxide,ATO)对人急性髓系白血病细胞KG-1的作用及可能机制。方法:CCK-8法检测普纳替尼及ATO对KG-1细胞增殖的影响;流式细胞术AnnexinⅤ/PI双重染色法检测细胞凋亡;实时荧光定量PCR检测细胞凋亡相关基因的表达;Western blot检测凋亡相关蛋白、成纤维细胞生长因子受体1(fibroblast growth factor receptor 1,FGFR1)蛋白及信号通路分子磷酸化水平的表达变化。结果:(1)ATO及普纳替尼对KG-1细胞的增殖抑制作用呈剂量依赖性,两药联合较单药作用具有更高的增殖抑制率、更少的集落形成及更多的细胞凋亡,差异均有统计学意义。(2)与DMSO组相比,ATO或普纳替尼均能显著下调Bcl-2表达,上调Bax及Caspase-3表达(P均<0.05);与单药作用相比,联合用药促进Bax及Caspase-3表达的作用更强(P均<0.01)。(3)普纳替尼显著抑制FGFR1基因及蛋白的表达(P均<0.01),ATO的加入并未使FGFR1表达进一步下降。信号通路研究显示,ATO可以显著抑制m-TOR和MAPK、STAT5的磷酸化(P均<0.001),但对PI3K/AKT、STAT3的磷酸化无明显影响。普纳替尼可以显著抑制FGFR1蛋白表达及STAT3、STAT5的磷酸化(P均<0.001),但对PI3K/AKT及MAPK的磷酸化无明显影响。两药联合后,STAT3的磷酸化水平较ATO或普纳替尼单药组进一步下调(P均<0.01)。结论:普纳替尼及ATO可能通过不同机制抑制KG-1细胞增殖及集落形成并诱导细胞凋亡;两药联合可进一步增强对KG-1细胞株的抑制效应。

Objective:To explore the effects and possible mechanisms of the arsenic trioxide(ATO)and small molecule tyrosine kinase inhibitor ponatinb on KG-1 cells in vitro.Methods:Effects of ATO and ponatinib on proliferation of KG-1 cells were detected by CCK-8,and the apoptosis was assessed by Annexin V-FITC.Reverse transcription quantitative polymerase chain reaction(q-PCR)analysis was used to detect the expression of apoptosis-related genes.Western blott was performed to explore the expression levels of apoptosis-related proteins,fibroblast growth factor receptor 1(FGFR1)and phosphorylated signal molecules.Results:①Both ATO and ponatinib effectively inhibited cell proliferation by dose dependent manners.The combination of the two drugs exhibited higher proliferation inhibition rate,less colony formation and more cell apoptosis compared to the single drug treatment.②Compared with the DMSO group,treatment with either ATO or ponatinib led to significant down-regulation of Bcl-2,up-regulation of Bax and Caspase-3(P<0.05).The combination of the two drugs up-regulated the expression of Bax and Caspase-3 more than single drug treatmen(t both P<0.01).③Punatinib significantly inhibited the expression of FGFR1 gene and protein(both P<0.01),and the addition of ATO did not decrease FGFR1 expression further.Signaling pathway studies showed that ATO significantly inhibited the phosphorylation of MAPK,m-TOR,and STAT5,but had no significant effect on the phosphorylation of PI3K/AKT and STAT3.Ponatinib markedly inhibited the phosphorylation of STAT3/5,and FGFR1 expression(both P<0.001),but had no significant effect on the phosphorylation of PI3K/AKT and MAPK.The phosphorylation level of STAT3 was further down-regulated by the combination of the two drugs compared with ATO or pratinib monotherapy(both P<0.01).Conclusion:ATO and ponatinib may inhibit KG-1 cell proliferation and colony formation and induce cell apoptosis through different mechanisms.The combination of the two drugs can further enhance the inhibitory effect on KG-1 cells.