四硫化四砷诱导K562细胞凋亡的机制研究

Apoptosis Mechanism in Human Chronic Myelogenous Leukemia K562 Cells Induced by Tetra-arsenic Tetra-sulfide

为了探讨四硫化四砷(As4S4)诱导慢性粒细胞性白血病(CML)细胞株K562凋亡及机制,用台盼蓝染色和MTS方法观察四硫化四砷对K562细胞生长抑制的影响,用瑞氏染色检测药物处理后的细胞形态学变化和细胞凋亡发生,流式细胞术检测细胞凋亡率和细胞周期改变,实时定量逆转录聚合酶链反应(realtimequantitativePCR)检测BCRABL、Bcl2、BclXL、Bad和Bax等mRNA的表达,Westernblot方法检测BCRABL、Akt和pAkt蛋白的表达。结果表明:四硫化四砷能明显抑制K562细胞活力,在0.5μmol/L浓度下培养24小时,细胞生长明显受抑制且有剂量和时间依赖关系。小于0.1μmol/L的四硫化四砷对K562细胞活力无明显影响。K562细胞经与四硫化四砷培养24至48小时后,出现典型的凋亡改变。与四硫化四砷培养12小时后,1.0μmol/L条件下,K562细胞凋亡率上升,大于2.0μmol/L浓度的四硫化四砷能明显诱导细胞凋亡,四硫化四砷能使K562细胞周期阻滞于G2/M期。四硫化四砷能使K562细胞BclXL的mRNA表达下调,但对Bcl2、Bad、Bax和BCRABL等的mRNA表达无影响。四硫化四砷能使K562细胞的pAkt蛋白表达下调,而BCRABL蛋白表达无变化。结论:四硫化四砷能有效地通过诱导凋亡抑制K562细胞生长,其机制可能与抑制凋亡途径因子BclX和pAkt等表达有关。

To explore the effects of tetra-arsenic tetra-sulfide （ AsaS4 ） in treatment of human chronic myelogenous leukemia K562 cells and its mechanism, trypan blue staining and microculture MTS assay were used to measure the effects of As4S4 on growth inhibition of K562 ceils; the morphologic change was determined by Wright＇s staining assay. The apoptosis rate and cell cycle were detected by flow cytometry; the changes of transcript and protein level were determined by real-time quantitative RT-PCR and Western blot analysis, respectively. The results indicated that As4S4 had signigicant cytotoxicity on K562 cells. At the concentration of 0.5μmol/L, the cell viability decreased significantly after being cultured with AsaS4 for 24 hours. When the concentration was lower than 0.1μmol/L, As4S4 had a little effect on K562 cells. The effect of As4S4 on K562 was time- and concentration- dependent. After being cultured with ASaS4 at the concentration of 1.0 Fmol/L for 24 to 48 hours, K562 cells displayed typical morphological changes of apoptosis. At a concentration greater than or equal to 1.0 μmol/L, ASaS4 could induce apoptosis significantly. After 12 hours of incubation with 1.0 μmol/L As4S4, the apoptosis rate increased from （3.47 ± 0.42）% to （6.16 ± 0.98% ）. At the same time, the percentage of cells in Gt phase decreased from （69.65 ± 3.24 ）% to （50.53 ± 2.86）%, whereas the percentage of cells in G2/M phase increased from （9.56 ± 2.51 ） % to （ 12.91 ± 2.13 ） %. The mRNA level of Bcl-XL and the protein level of pAkt were down-regulated after the inhibition of As4 S4, while the mRNA expression of Bcl-2, Bad and Bax had no change. Both of the transcript and protein level of bcr-abl had no change after incubation with As4 S4. It is concluded that ASaS4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4 S4 interferes with pAkt pathway and down-regulates Bcl-XL, which may be involved in the response of K562 to this agent.