丙戊酸联合伏立诺他增强阿糖胞苷对人Dami白血病细胞株杀伤作用的机制研究

Mechanisms of synergistic antileukemia effect of cytarabine combined with valproic acid and suberoylanilide hydroxamic acid

目的探讨丙戊酸(VPA)联合伏立诺他(SAHA)增强阿糖胞苷(Ara-c)对人Dami白血病细胞株的杀伤作用,并探讨其分子学机制。方法设空白对照组、Ara-c组、VPA组、SAHA组、VPA+Ara-c组、SAHA+Ara-c组,VPA+SAHA+Ara-c组,分别作用于对数生长期的Dami白血病细胞,MTT比色法检测药物对细胞的生长抑制作用;用PI法通过流式细胞仪检测细胞周期;RT-PCR方法检测转录因子GATA-1、胞苷脱氨酶(CDA)及脱氧胞苷激酶(DCK)mRNA水平的变化。结果 VPA和SAHA共同联合Ara-c用药组细胞生长抑制率明显高于VPA联合Ara-c组及SAHA联合Ara-c组,各组间相比,具有显著性差异。Dami细胞经过VPA和SAHA处理后均出现细胞周期阻滞现象,主要阻滞在G1期,与对照组相比差异有显著性(P<0.05);VPA和SAHA联合用药组G1期细胞所占比例高于SAHA组(P<0.05),与VPA组差异无显著性(P>0.05)。VPA组、SAHA组、VPA和SAHA联合用药组GATA-1和CDA mRNA表达水平均低于对照组(P<0.05),各组间差异无显著性(P>0.05);VPA组DCK mRNA表达水平高对照组(P<0.05)、SAHA组与对照组比较差异无显著性(P>0.05)、VPA和SAHA联合用药组DCK mRNA表达水平高于VPA组和SAHA组(P<0.05)。结论 VPA联合SAHA可增强阿糖胞苷对Dami白血病细胞的杀伤作用,可能是通过增加DCK的表达,从而增加阿糖胞苷体内活性代谢产物所致。

Objective To explore the mechanisms of synergistic antileukemia effect of cytarabine combined with Valproic acid （VPA） and suberoylanilide hydroxamic acid （SAHA）. Methods Dami cells were co-treated with SAHA, VPA and cytarabine. Cell growth was measured by MT-F assay. Cell cycle distribution was determined using Propidium iodide （piodize, PI ） single staining and flow cytometric analysis. Changes of mRNA level of transcription factors including GATA-1, CDA and DCK were detected by real-time quantitative RT-PCR method. Results SAHA and VPA had synergistic cytotoxicity against leukemia Dami cells in combination with cytarabine . The cell cycle were blocked after VPA and SAHA treatment with most of the cells blocked in G1 phase. Compared with the controlgroup, the proportion of cells blocked increased significantly in G1 phase in every dose group （P 〈 0. 05）. Compared with SAHA group, the proportion of cells blocked increased significantly in G1 phase （P 〈0.05） in the VPA combined with SAHA group. The CDA,GATA-1 mRNA level of the VPA group, the SAHA group and the combination group were lower than the control group （P 〈 0.05 ）. The DCK mRNA level in VPA group was higher than the control group （P 〈 0.05 ）. Whereas the SAHA group showed no significant difference with the control group （ P 〉 0.05 ）. The DCK mRNA level in combination group was higher than that in the VPA group and the SAHA group （ P 〈 0.05 ）. But the CDA,GATA-1 mRNA level had no significant difference （P 〉 0.05）. Conclusions The synergistic effect of cytarabine combined with Valproic acid （VPA） and suberoylanilide hydroxamic acid （SAHA） could be associated with the increased transcription of DCK, which then increase the amount of bioactive metabolites of cytarabine and thus enhance the antileukemic effect.