摘要
目的从分子水平探讨丙戊酸(VPA)诱导白血病细胞凋亡的机制,为临床治疗白血病提供理论依据。方法将U937、Jurkat clone E6-1(Jurkat)、BALL-1分别分为1mmol/LVPA组、1mmol/LVPA+1μmol/LzVAD-fmk组(zVAD为N-苯甲基氧化炭酰-缬氨酸-丙氨酸-天冬氨酸)和空白对照组,作用72h后在流式细胞仪上检测细胞凋亡;采用流式细胞仪检测各组细胞中Bcl-2、Bax和Bcl-xl的平均荧光指数(MFI)以及胱冬肽酶(caspase)3、8和9的含量;Western-blotting检测各组作用前后P44/42有丝分裂原激活蛋白激酶(MAPK)表达及磷酸化水平。结果1mmol/LVPA诱导U937和Jurkat的凋亡率分别为(75·8±4·2)%和(53·5±5·9)%,与对照组比较,P<0·01;多caspase抑制剂zVAD-fmk可全部抑制U937凋亡,凋亡率为(2·9±0·4)%;可部分抑制Jurkat凋亡,凋亡率下降至(15·4±1·4)%,与1mmol/LVPA组比较,P<0·01。1mmol/LVPA未能诱导BALL-1凋亡(P>0·05)。VPA对3种细胞内Bcl-2、Bax和Bcl-xl无明显影响。VPA作用后U937中caspase-3水平由(14·09±1·19)%上升至(32·30±2·47)%,caspase-8水平由(4·58±1·41)%上升至(86·47±3·26)%,均P<0·01,caspase-9变化不显著。Jurkat中caspase-3由(12·01±1·63)%上升至(35·56±0·27)%,caspase-9由(13·89±1·71)%上升至(75·89±4·08)%,均P<0·01,caspase-8变化不显著。BALL-1中caspase-3略有减少,P<0·05。VPA作用后3种细胞均表现为P44/42MAPK蛋白总量及磷酸化水平下降,P<0·01。结论VPA通过激活caspase-3和caspase-8诱导U937凋亡;诱导Jurkat凋亡时涉及caspase-3、9的活化;抑制P44/42MAPK通路是VPA诱导凋亡的另一机制。VPA并非通过改变细胞中Bcl-2等的含量造成上述细胞凋亡。高表达Bcl-2可对抗VPA的作用。
Objective To overcome the drug-resistance of tumor cells is one of the methods of improving the therapeutic results. Histone deacetylase inhibitors （HDACIs） is a novel class of chemotherapeutic agents which can induce apoptosis of tumor cells. Valproic acid（VPA） is a common drug used in the treatment of epilepsy. It has been shown that VPA has a marked HDACIs effect at the pharmaceutical level, and can induce the differentiation and apoptosis of transformed cells. But the mechanism of its effect has not been clarified. The aim of this study was to investigate the mechanism of VPA in inducing the apoptosis of leukemic cells at molecular level. Methods The cell lines U937, Jurkat clone E6-1 （Jurkat） and BALL-1 were cultured in RPMI 1640 medium containing 20% calf serum, then divided into three groups （control group, 1 mmol/L VPA group and 1 mmol/L VPA ＋ 1 μmol/L Pan-caspase inhibitor zVAD-fmk group）. At 72 hours after the treatment, the cells were double stained with Aunexin and PI （propidium iodide） and then were analyzed with the flow cytometry （FCM） to detect apoptosis. Before and after treatment with VPA the mean fluorescence index （MFI） of Bcl-2, Bax, Bcl-xl and the levels of caspase 8, 9 and 3 were also detected with the FCM. The changes of P^44/42 mitogen activating protein kinase （MAPK） and phosphorylated P^44/42 MAPK were determined by Western blotting. Results Seventy-two hours after the treatment, 1 mmol/L VPA induced apoptosis of U937 and Jurkat. The apoptotic rate of U937 was（75.78 ±4. 20）% and that of Jurkat was（53.50 ±5.87）% （P 〈0.01, vs. coutrol group） ; zVAD-fmk could fully inhibit the apeptosis of U937, and the apoptotic rate was （2. 89 ±0. 36）% ; while it could partly inhibit the apeptosis of Jurkat, and the apoptotic rate was （ 15.38 ± 1.40） % （ P 〈 0. 01 ）. 1 mmol/L VPA could not induce the apoptosis of BALL-1 which had a high expression level of Bcl-2. The MFI of Bcl-2, Bax and Bcl-xl in these three cell lines did not change significantly with VPA （P 〉 0. 05）. After treatment with VPA, the level of caspase 3 in U937 increased from （ 14. 09 ± 1.19） % to （ 32.30 ± 2.47 ） % , and caspase 8 from （4. 58 ± 1.41 ） % to （ 86.47 ±3.26 ） % （ P 〈 0.01 ） , but there was no significant change in caspase 9 [ （ 13.25 ±3.11）% and （10.95 ± 1. 30）% ]. In Jurkat, the level of caspase 3 increased from（ 12. 01 ± 1.63 ） % to （ 35. 56 ± 0. 27 ） % , and caspase 9 from （ 13. 89 ±1.71 ） % to （ 75. 89 ± 4. 08 ） % （ P 〈 0. 01 for both）; no significant change was observed for caspase 8 [ （5.94 ± 1.38）% and （5.44 ±0.72）% ]. In BALL-1, there was a slight decline in caspase 3 （P 〈0. 05）. With the effect of VPA, levels of P^44/42MAPK and phosphorylated P^44/42 MAPK decreased in all three cell lines （ P 〈 0.01 ）. Conclusion VPA could induce apoptosis of U937 through the activation of caspase 3 and 8 ; and it induced the apoptosis of Jurkat involving the activation of caspase 3 and 9. P^44/42MAPK pathway also plays an important role in this course. VPA induced apoptosis of these cell lines without the alteration of Bcl-2, Bax and Bcl-xl. High level of Bcl- 2 could antagonize the effect of VPA.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2005年第12期894-898,共5页
Chinese Journal of Pediatrics
