Xue Hong-man, Li Wen-yi, Guo Hai-xia, Xia Yan, Chen Qin, Liu Yong
Department of Pediatrics, Second Affiliated Hospital, Zhongshan University, Guangzhou 510120, China.
Zhonghua Er Ke Za Zhi. 2005 Dec;43(12):894-8.
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.
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 micromol/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.
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. control group); zVAD-fmk could fully inhibit the apoptosis of U937, and the apoptotic rate was (2.89 +/- 0.36)%; while it could partly inhibit the apoptosis 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(44/42) MAPK and phosphorylated P(44/42) MAPK decreased in all three cell lines (P < 0.01).
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(44/42) MAPK 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.
克服肿瘤细胞的耐药性是提高治疗效果的方法之一。组蛋白去乙酰化酶抑制剂（HDACIs）是一类新型化疗药物，可诱导肿瘤细胞凋亡。丙戊酸（VPA）是治疗癫痫的常用药物。研究表明，VPA在药理水平上具有显著的HDACIs作用，可诱导转化细胞分化和凋亡。但其作用机制尚未阐明。本研究旨在从分子水平探讨VPA诱导白血病细胞凋亡的机制。
将U937、Jurkat克隆E6 - 1（Jurkat）和BALL-1细胞系培养于含20%小牛血清的RPMI 1640培养基中，然后分为三组（对照组、1 mmol/L VPA组和1 mmol/L VPA + 1 μmol/L泛半胱天冬酶抑制剂zVAD-fmk组）。处理72小时后，用Annexin和PI（碘化丙啶）对细胞进行双染，然后用流式细胞术（FCM）分析检测细胞凋亡。用FCM检测VPA处理前后Bcl-2、Bax、Bcl-xl的平均荧光指数（MFI）以及半胱天冬酶8、9和3的水平。通过蛋白质免疫印迹法测定P(44/42)丝裂原活化蛋白激酶（MAPK）和磷酸化P(44/42) MAPK的变化。
处理72小时后，1 mmol/L VPA诱导U937和Jurkat细胞凋亡。U937细胞凋亡率为（75.78±4.20）%，Jurkat细胞凋亡率为（53.50±5.87）%（P < 0.01，与对照组相比）；zVAD-fmk可完全抑制U937细胞凋亡，凋亡率为（2.89±0.36）%；而对Jurkat细胞凋亡有部分抑制作用，凋亡率为（15.38±1.40）%（P < 0.01）。1 mmol/L VPA不能诱导Bcl-2高表达的BALL-1细胞凋亡。这三种细胞系中Bcl-2、Bax和Bcl-xl的MFI随VPA处理无明显变化（P > 0.05）。VPA处理后，U937细胞中半胱天冬酶3水平从（14.09±1.19）%升至（32.30±2.47）%，半胱天冬酶8从（4.58±1.41）%升至（86.47±3.26）%（P < 0.01），但半胱天冬酶9无明显变化[（13.25±3.11）%和（10.95±1.30）%]。在Jurkat细胞中，半胱天冬酶3水平从（12.01±1.63）%升至（35.56±0.27）%，半胱天冬酶9从（13.89±1.71）%升至（75.89±4.08）%（两者均P < 0.01）；半胱天冬酶8无明显变化[（5.94±1.38）%和（5.44±0.72）%]。在BALL-1细胞中，半胱天冬酶3略有下降（P < 0.05）。随着VPA的作用，三种细胞系中P(44/42) MAPK和磷酸化P(44/42) MAPK水平均下降（P < 0.01）。
VPA可通过激活半胱天冬酶3和8诱导U937细胞凋亡；通过激活半胱天冬酶3和9诱导Jurkat细胞凋亡。P(44/42) MAPK途径在这一过程中也起重要作用。VPA诱导这些细胞系凋亡时，Bcl-2、Bax和Bcl-xl无改变。Bcl-2高水平可拮抗VPA的作用。
