低浓度雷公藤甲素联合伊达比星诱导急性髓系白血病干细胞凋亡及其机制

Low-dose Triptolide in Combination with Idarubicin Induce Apoptosis of Acute Myeloid Leukemia Stem Cells Via Modulation of HIF-1α Pathways

【目的】探讨低浓度雷公藤甲素联合伊达比星(IDA)在体外对急性髓系白血病细胞(AML)干细胞凋亡的影响及其分子机制。【方法】应用流式细胞仪分选KG1a细胞中具有AML干细胞特性的CD34+CD38-亚群;对分选的CD34+CD38-KG-1α细胞进行免疫表型和细胞周期的测定;流式细胞术检测IC20浓度的雷公藤甲素、IC50浓度的IDA单药及上述浓度的雷公藤甲素联合IDA对CD34+CD38-KG1a细胞的诱导凋亡作用;蛋白质印迹法检测药物处理后HIF-1a及其下游CXCR4、VLA-4蛋白表达水平的变化。【结果】分选后CD34+CD38-亚群细胞占KG-lα细胞比例达(98.15±1.64)%,,细胞周期检测显示G0/G1期细胞比例达(82.40±3.82)%。空白对照组、IC20浓度的雷公藤甲素(5 nmol/L)单药组、IC50浓度的IDA(27nmol/L)单药组及上述浓度的雷公藤甲素联合IDA组作用于AML干细胞细胞,24 h凋亡率分别为:(5.63±0.67)%、(9.11±0.33)%、(24.85±1.70)%和(76.87±8.34)%,联合组诱导AML干细胞凋亡比较显著高于雷公藤甲素单药组及IDA单药组(均P<0.001)。蛋白质印迹法结果显示单药组及联合组均可以不同程度下调AML干细胞HIF-1a、CXCR4及VLA-4蛋白的表达水平,其中联合组最为明显。【结论】低浓度雷公藤甲素可显著提高IDA对AML干细胞诱导凋亡作用,其机制可能是通过抑制HIF-1α及其下游通路实现的。

[Objective] To investigate the effect of low-dose triptolide （TRI） in combination with idarubicin （IDA） on acute myeloid leukemia stem cells and the relationship with HIF-1α pathway.[Methods] CD34^＋CD38^-KG1a cells was sorted from KG1a cell lines by fluorence-activated cell sorting （FACS） analysis.Immunophenotype and cell cycle analysis of CD34^＋CD38^-KG1a cells were analyzed by fluorescence-activated cell sorting analysis.Annexin-V/PI double staining was used to detect the effects of low-dose TRI in combination with IDA on apoptosis of CD34^＋CD38^-KG1a cells.Western bloting to analyze the expression of HIF-1α and its downstream protein CXCR4,VLA-4 in CD34^＋CD38^-KG1a cells after treatment with TRI,IDA,and TRI＋IDA.[Results] After sorting,98.15-± 1.64% of the cells were labeled for CD34^＋CD38^-.Cell cycle analysis also showed that proportion of G0/G1 cells was 82.4 ± 3.82%.CD34^＋CD38^-KG1a cells were exposed to 5 nmol/L TPL,27 nmol/L IDA and TRI±IDA for 24 h.The apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis.Apoptotic ratio of cells treated by IDA with TPL was significantly increased compared to cells treated by IDA alone （24.85 ± 1.70% vs.76.87 ± 8.34%,P 〈 0.001）.Western blot results showed markedly decrease the expression of HIF-1α,CXCR4 and VLA-4 in CD34^＋CD38^-KG1a cells treated with low-dose TRI in combination with IDA.[Conclusion] A relatively low concentration of TPL in combination with IDA could induce apoptosis of AML stem cells in vitro via inhibition of HIF-1α pathway.