大蒜提取物Z-ajoene对U937细胞增殖抑制作用机制的研究

Mechanism of growth inhibition of Z-ajoene on U937 cells

目的 研究大蒜含硫化合物Z ajoene抗肿瘤作用的分子机制。方法 应用MTT法测定Z ajoene对不同白血病细胞株的生长抑制作用。采用流式细胞技术 (FACS)分析Z ajoene对U937细胞周期的影响以及对该细胞的诱导凋亡作用。用Western blot的方法检测Z ajoene对凋亡相关蛋白以及微管蛋白表达的影响。结果 Z ajoene对两种白血病细胞系HL6 0和U937有着相近的生长抑制作用 ,IC50 值均约为10 μmol·L-1。细胞周期分析发现 ,在给药初期和药物浓度较低时G2 /M期细胞数量明显升高 ;2 0 μmol·L-1Z ajoene作用 2 4h时 ,G2 /M期细胞比对照组升高约15 2 5 %。Z ajoene可以诱导U937细胞凋亡 ,并呈浓度和时间依赖性。实验结果表明 ,细胞凋亡过程中的关键蛋白酶caspase 3的激活及其底物PARP蛋白的裂解随着给药浓度和时间的增加而增加。此外 ,Z ajoene也产生以出现 2 3ku裂解带为特征的Bcl 2裂解。另外 ,实验浓度的Z ajoene对β 微管蛋白的表达有抑制作用 ,而对α 微管蛋白表达无明显改变。结论 Z ajoene能明显抑制U937细胞株的生长并可阻断U937细胞周期于G2 /M期 ,且这种阻断早于细胞凋亡。Z ajoene抗肿瘤机制与诱导细胞凋亡和干扰微管蛋白聚合相关。

AIM To study the antitumor mechanism of Z-ajoene, a simple natural compound extracted from garlic. METHODS Cell growth inhibition in vitro was evaluated with MTT assay in floating U937 cells compared with HL60 cells. Z-ajoene-induced cell cycle block was investigated using flow cytometry assay. Western blot analysis was applied to determine modification of certain proteins related to apoptosis and tubulins. RESULTS Z-ajoene displayed quite similar growth inhibition ability on two leukemia cell lines U937 and HL60 with the similar IC 50 values of 10 μmol·L -1. Flow cytometry analysis indicated that Z-ajoene induced cell accumulation in the G 2/M phase of the cell cycle. Following a treatment of 20 μmol·L -1 Z-ajoene for 24 h, the percentage of cells arrested at G 2/M phase was 152.5% higher than that of controls. Moreover, Z-ajoene induced apoptosis of U937 cells. The activation of procaspase-3 (Cpp32) and PARP cleavage were detectable with a time and dose-dependent manner, after exposure to Z-ajoene by means of Western blotting. Furthermore, incubation of U937 cells with Z-ajoene induced Bcl-2 cleavage characterized by the appearance of a 23 ku fragment. In addition, the data showed that β-tubulin was degraded after addition of Z-ajoene. However, there was no change of α-tubulin expression during the procedure of Z-ajoene treatment. CONCLUSION Z-ajoene exhibites high abilities to inhibit U937 cell growth, blocking the cell cycle at G 2/M phase and inducing programmed cell death. Mitotic blocking causd by Z-ajoene appeared earlier than apoptosis. All these findings suggest that antitumor mechanism of Z-ajoene is related to its actions of apoptosis induction and interruption of tubulin assembly.