组蛋白去乙酰化酶抑制剂MS-275对脑胶质瘤细胞增殖的影响

目的探讨组蛋白去乙酰化酶抑制剂MS-275对脑胶质瘤细胞系U251细胞增殖的影响。方法应用不同浓度的MS-275作用于U251细胞,分别培养24、48和72h,采用细胞计数试剂盒检测其对U251细胞增殖的抑制作用,瑞氏-姬姆萨染色观察细胞形态变化,碘化丙啶单染法检测细胞周期变化,膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染法经流式细胞仪检测细胞凋亡率,Western-blot检测聚腺苷二磷酸核糖聚合酶(PARP)经天冬氨酸特异性半胱氨酸蛋白酶(caspase)3作用后的裂解产物。结果 MS-275对U251细胞增殖的抑制作用在相同作用时间下随药物浓度的增加而增大,在同一浓度作用下随作用时间延长而增大,其最佳浓度为10nmol/L。MS-275作用后U251细胞呈现凋亡,胞质和胞核浓缩或碎裂。10nmol/mlMS-275分别作用24、48和72h后细胞凋亡率分别为(2.1±0.4)%、(11.7±0.5)%和(29.0±2.3)%,三者差异显著(P<0.05);随作用时间延长,G1期细胞所占比例逐渐减少,G2期细胞所占比例先增多后减少,药物作用24h所占比例最高,S期细胞所占比例变化不明显,作用48h后开始出现亚G0凋亡峰,且逐渐增大,而对照组没有出现该峰;PARP被caspase3剪切。结论 MS-275对胶质瘤细胞的增殖抑制作用具有浓度和时间双重依赖性;这种作用是通过激活caspase3信号通路诱导细胞凋亡实现的。

Effect of histone deacetyiase inhibitor, MS-275 on proliferation of brain glioma cells

Objective To investigate the effect of a histone deacetylase inhibitor,MS-275 on the proliferation of brain glioma cells.Methods Human brain glioma cell lines,U251 cells were cultured respectively for 24,48 and 72 hours in the media containing different concentrations of MS-275.The proliferation of U251 cells was determined by cell counting kit 8.The change in the cellular morphology was observed by Wright-Giemsa staining.The cell cycle was analyzed by propidium iodie （PI） dyeing and flow cytometry.The rate of apoptosis was detected by annexin V-fluoresceisothiocyanate and PI dyeing and flow cytometry.The expression of the protein of poly ADP ribose polymerase （PARP） were detected by Western blot.Results The effect of MS-275 on the inhibition of U251 cells proliferation was concentration-and time-dependent.Apoptosis of U251 cells was observed,and U251 cells cytoplasm and nuclei were enriched or broken up after MS-275 treatment.The proportions of G 0/G 1 phase and the G 2/M cells to all the cells were significantly decreased compared to the control groups （P 0.01 or 0.05）.The apoptosis peak appeared before the G 1 phase in the U251 cells 48 hours after the treatment with MS-275.The apoptosis rates of U251 cells cultured in the medium containing 10 nmol/ml MS-257 were （2.133± 0.416）%,（11.667±0.503）% and （29.000±2.307）% respectively 24,48 and 72 hours after the cultures.There were significant differences in the apoptosis rate among three groups （P 0.05）.The apoptosis rates were significantly higher 48 and 72 hours after the culture with 10 nmol/ml MS-257 than that in the control group （P 0.05）.The cleavage of PARP were detected by Western blot in the U251 cells 48 and 72 hours the culture with MS-257.Conclusions The proliferation of U251 cells in a time and dose-dependent manner can be inhibited by MA-275.The inhibitory effect of MS-257 on U251 cells proliferation is achieved by activation of cysteine-dependent aspartate-specific proteases 3 signaling pathway inducing apoptosis.