雷帕霉素联合5-氮-2’-脱氧胞苷对急性白血病Nalm-6细胞生长抑制作用及其机制研究

Inhibitory effect of combination of rapamycin and 5-Aza-dC on Nalm-6 cells and the underlying mechanisms

目的探讨雷帕霉素（RAPA）联合5-氮-2＇-脱氧胞苷（5-Aza-dC）对急性白血病Nalm-6细胞增殖和凋亡的影响及其作用机制。方法培养复苏后的Nalm-6细胞至对数生长期，采用不同浓度的RAPA、5-Aza-dC单药及两药联用处理Nalm-6细胞。CCK-8比色法检测细胞增殖，Annexin V-PE／7-AAD标记及流式细胞术检测细胞凋亡，Western blot检测细胞TSC2及磷酸化哺乳动物雷帕霉素靶蛋白（mTOR）的表达。结果RAPA及5-Aza-dC均能抑制Nalm-6细胞增殖和诱导Nalm-6细胞凋亡（P〈0．05），RAPA联合5-Aza-dC对Nalm-6细胞的增殖抑制和凋亡诱导具有协同作用（P〈0．05）。5-Aza-dC上调Nalm-6细胞的TSC2蛋白表达，RAPA及5-Aza-dC降低mTOR的磷酸化水平，RAPA联合5-Aza-dC可提高对mTOR磷酸化的抑制。结论RAPA联合5-Aza-dC可增强对Nalm-6细胞增殖的抑制作用及凋亡的诱导作用，作用机制可能是抑制了mTOR的磷酸化。

Objective To investigate the effects and underlying mechanisms of combination of mTOR inhibitor rapamycin （RAPA） and DNA methyltransferase inhibitor 5-Aza-2＇-deoxycytidine （5-Aza-dC） on the cell proliferation and apoptosis of human acute leukemia cell lines Nalm-6 for providing a new promising targeted therapeutic strategy for acute leukemia. Methods The recovered Nalm-6 ceils were cultured to exponential phase, and divided into groups in which RAPA and 5-Aza-dC with different concentrations or their combinations. The effects on cell proliferation were analyzed by CCK-8 assay. Apoptosis was detected by flow cytometry with Annexin V-PE/7-AAD staining. Western blot analysis was performed to examine the expression of TSC2 protein and phosphorylation of mTOR. Results RAPA and 5-Aza-dC could inhibit the proliferative activity of Nalm-6 cells in a dose dependent manner （P 〈 0.05）. Combination of 5-Aza-dC with RAPA exerted a synergistic anti-poliferative effect on Nalm-6 cells （CI 〈 1）. apoptosis was induced by RAPA and 5-Aza-dC in Nalm-6 cells （P 〈 0.05）. Compared with RAPA alone groups, combined groups could observably increase the apoptosis rate of Nalm-6 ceils （P 〈 0.05）. In Nalm-6 cells, 5-Aza-dC could increase the expression of TSC2 protein. Though RAPA and 5-Aza-dC individually inhibited the phosphorylation of mTOR, combination of them were more significantly effective in inhibiting mTOR phosphorylation. Conclusions Combination of RAPA and 5-Aza-dC exerted a synergistic effect on inhibition of cell proliferation and induction of cell apoptosis in Nalm-6 ceils. The mechanisms may be associated with the regulation of the proteins expression of roTOR pathway.