低剂量地西他滨联合伊马替尼对K562细胞的增殖抑制作用

Effect of low-dose decitabine in combination with imatinib mesylate in K562 cells

目的研究低剂量地西他滨（DAC）联合伊马替尼（IM）对K562细胞株的增殖抑制作用及对bcr-abl表达的影响。方法单药及两药联合后，通过四甲基偶氮唑蓝（MTT）法观察药物对K562细胞株的增殖抑制作用，流式细胞术检测药物对K562细胞株早期凋亡率及细胞周期，巢式反转录-聚合酶链反应（RT-PCR）半定量检测药物对K562细胞株bcr—ablmRNA表达。结果DAC与1M单药对K562细胞的抑制作用呈浓度时间依赖性。两药联合用药抑制作用较单药组明显（F=43．947、165．580、321．193、296．101，均P〈0．05），24、48、72h各浓度组与对照组比较差异均有统计学意义（F=202．759、168．457、417．538，均P〈0．05）。DAC及IM单药作用药物对K562细胞株均使G．期细胞明显增多，IM0．2μmnl／L作用于K562细胞株48h可见6．7％早期凋亡细胞，IM0．2μmol／L联合DAC4μmol／L早期凋亡细胞增加至8．4％。bcr—ahlmRNA表达水平降低，DAC4μmol／L作用48h后可降低K562细胞中bcr-ablmRNA表达（约14％），IM0．2μmol／L降低约40％，联合用药表达量明显降低（约60％）。联合用药组与单药组比较差异有统计学意义（F=71．981，P〈0．05）。结论DAC对K562细胞的增殖抑制作用与细胞周期阻滞、诱导凋亡及降低bcr-ablmRNA表达有关，两药联合可显著抑制K562细胞增殖。

Objective Imatinib mesylate （IM） is the most active agent in treating chronic myeloid leukemia （CML）. 5-Aza-2-deoxyeytidine （DAC） is a cytosine analogue that inhibits DNA methylation and the activity in myeloid leukemia. Therefore, we investigated combining these two drugs in human leukemia cell line K562. Methods The effects of IM and DAC was examined in K562 cells including cell viability using MTT method, cell cycle phase and cell death using flow cytometric （FCM）, and the expression of bcr-abl mRNA by RT-PCR. Results Both DAC and IM resulted in time and concentration-dependent induction of cell death. DAC and IM in combination produced a greater inhibition of growth against K562 cells （F =43.947, 165.580, 321.193, 296.101, P〈0.05）. The main effect and interaction between two drugs was statistically significant （F = 202.759, 168.457, 417.538, P 〈0.001） after 24 h, 48 h, 72 h and a greater reduction in expression of bcr-abl mRNA than either agent alone. The difference was statistically significant （F =71.981, P 〈0.05）. The number of G2 phase cells were increased significantly when induced by single agent. 48 h incubation with IM 0.2 Ixmol/L alone or combined with DAC 4 μmol/L showed 6.7 %, 8.4 % pre-apoptosis cells, respectively. After incubation for 48 h with DAC 4 μmol/L, the expression of mRNA were decreased by 14 %, IM 0.2 μmol/L showed 40 % reduction, and combination group were significantly depressed for the mRNA expression by 60 %. Conclusion The combination of DAC and IM showed synergistic effects on cell death in K562 cells. These data suggested that DAC used in combination with IM has clinical potential in the treatment of chronic myeloid leukemia.