AKT/ROS信号通路对急性髓细胞白血病增殖的影响及靶向作用研究

AKT signal transduction pathway is constitutively activated and cellular reactive oxygen species (ROS) are highly expressed in acute myeloid leukemia (AML) blasts, correlated with an unfavorable prognosis in this disease. It has been demonstrated in recent studies that a positive interplay existed between ROS and AKT signaling pathways in leukemia, conferring to development of leukemia and promoting proliferation of AML blasts. Our previous published data indicated that ROS was required in maintaining normal hematopoietic stem cell (HSC) pool. In our pilot study, intracellular ROS level was identified to be higher in AML leukemic stem cells (LSC) than in normal counterpart. Antioxidant NAC resulted in reduction of cellular ROS level, coincided with inhibition of proliferation in K562 leukemic cell line that overexpressed BCR-ABL-driven AKT signaling transduction pathway. In order to target AKT/ROS signaling transduction pathways in AML, AKT inhibitor (AKTi-1/2) in combination with antioxidant NAC will be applied in AML blasts on the basis of our previous data. By performing multiparametric intracellular flow cytometry, cell proliferation assay and colony-forming cells assay, we will analyze AKT/ROS expression in dissected AML LSC and progenitor cell fractions, investigate the inhibitory effect of AKTi-1/2 with NAC on leukemic cell proliferation, and get a deeper insight into mechanism of increased ROS level in AML. Our project is expected to supply significant data regarding targeting treatment and improvement of the therapeutic results in AML.

AKT信号通路及细胞内活性氧（ROS）高水平表达于急性髓细胞白血病（AML），研究发现二者之间存在相互作用，促进AML发生和白血病细胞增殖。我们前期的研究证实了ROS在维持正常干细胞功能中发挥关键作用。预实验发现在AML白血病干细胞（LSC）中，ROS表达水平高于正常干细胞。在持续表达AKT信号通路的K562白血病细胞系中，抗氧化剂NAC能够下调ROS表达，抑制细胞增殖。为了揭示AKT/ROS信号通路在AML中的靶向作用，本项目拟在已有研究基础上，联合应用AKT抑制剂AKTi-1/2和抗氧化剂NAC处理细胞，通过多参数流式细胞分析，细胞增殖，克隆形成试验等方法，分析AKT/ROS信号通路在白血病干细胞和祖细胞群中的表达水平，探讨靶向作用AKT/ROS信号通路对AML增殖的影响及ROS增加的相关机制。本项目可为AML靶向治疗提供理论和实践依据，有利于进一步改善AML疗效。

对急性髓细胞白血病（AML）患者而言，虽然约60%的患者经常规化疗后可获得完全缓解，但长期无病生存率却极低。已有研究表明，AML存在AKT信号通路的组成性激活及细胞内活性氧簇（ROS）的高度表达，且二者间存在相互作用，可促进AML的发生和白血病细胞的增殖，这些均与疾病的不良预后相关。为了进一步揭示AKT/ROS信号通路在AML中的靶向作用，本研究应用了AKT抑制剂（AKTi1/2和NVP-BEZ235）及抗氧化剂（NAC）处理细胞，并通过多参数流式细胞术、细胞增殖及克隆形成试验等方法，分析了AKT/ROS信号通路在白血病干细胞和祖细胞群中的表达水平，探讨了靶向作用 AKT/ROS 信号通路对AML增殖的影响及ROS增加的相关机制。结果表明，在多种AML细胞株中，经AKT抑制剂和抗氧化剂处理后，细胞的AKT和ROS水平明显降低；与DMSO对照组相比，单药治疗组均可显著抑制细胞增殖并诱导细胞凋亡（P<0.05）；与单药治疗组相比，联合抑制AKT信号通路和ROS信号通路在抑制细胞增殖（MOLM-13, P<0.05; HL-60, P<0.05）和诱导细胞凋亡(MOLM-13, P<0.05; U937, P<0.05)中显示出协同效应。虽然在AML原代细胞的处理结果中，未见上述的联合效应，但中和ROS却可显著地损伤干祖细胞的克隆形成能力。此外，在实验中的所有AML原代细胞中，NAC可显著诱导细胞凋亡，且在多数AML原代细胞中可见AKTi1/2和NAC具有协同作用的趋势。上述研究结果揭示了AKT和ROS信号通路在AML中的可能发病机制，并为AML的靶向治疗提供了理论基础和实践依据，或许可为AML的治疗提供一种新的治疗策略。