辛二酰苯胺氧肟酸对人脐静脉内皮细胞增殖及血管形成的影响

Effect of suberoylanilide hydroxamic acid on proliferation and angiogenesis of human umbilical vein endothelial cells

目的探讨组蛋白去乙酰化酶抑制剂(Histone deacetylases inhibitor,HDACi)辛二酰苯胺氧肟酸(Suberoy-lanilide hydroxamic acid,SAHA)对人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)增殖及血管形成能力的影响。方法收集处于对数生长期的HUVEC,以不同浓度的SAHA分别处理24和48 h,另设不加SAHA的对照组,采用CCK-8法检测细胞的增殖活力,并计算增殖抑制率和半数抑制浓度(IC50)。采用流式细胞术检测经15μmol/L SAHA处理48 h的HUVEC凋亡和细胞周期,基质胶体外血管生成试验检测HUVEC的体外成管能力,Western blot法检测HUVEC细胞周期及凋亡相关蛋白的表达水平。结果随着SAHA浓度的增加及作用时间的延长,其对HUVEC增殖的抑制作用增强,SAHA浓度高于80μmol/L时,抑制率增加不明显,24和48 h的IC50值分别为60.53和30.49μmol/L;经15μmol/L SAHA处理48 h,与对照组相比,HUVEC的凋亡率明显增加(P<0.001),S期细胞比例明显升高(P<0.001),G0/G1期比例明显降低(P<0.001),体外成管能力明显下降,P21、caspase-3激活型、caspase-9酶原和激活型蛋白的表达水平均明显升高(P<0.001)。结论 SAHA能够抑制HUVEC增殖及体外血管形成能力,并使P21、caspase-3和caspase-9蛋白水平上调,为肿瘤的治疗提供了一种新的思路。

Objective To investigate the effect of suberoylanilide hydroxamic acid （SAHA）, a histone deacetylases inhibitor （HDACi）, on the proliferation and angiogenesis of human umbilical vein endothelial cells （HUVECs） in vitro. Methods HUVECs in logarithmic growth phase were collected and treated with SAHA at various concentrations for 24 and 48 h separately, using those untreated as control. The proliferative activity of HUVECs was determined by CCK-8 method, based on which the proliferation-inhibiting rate and median inhibitory concentration （IC50） were calculated. The apoptosis and cell cycle of HUVECs after treatment with 15 ~mol/L SAHA for 48 h were determined by flow cytometry. The angiogenesis in vitro of HUVECs was determined by Matrigel angiogenesis assay in vitro. The expression level of protein associated with cell cycle and apoptosis was determined by Western blot. Results SAHA showed dose- and time-dependent inhibitory effect on he proliferation of HUVECs. However, the inhibiting rate showed no significant increase when the SAHA concentration was more than 80 μmol / L. The ICs0 of SAHA after treatment for 24 and 48 h were 60. 53 and 30. 49 μmol/L, respectively. The apoptosis rate and percentage in S phase of HUVECs 48 h after treatment with 15 μmol/L SAHA increased significantly as compared with that in control group （both P 〈 0. 001 ）, while the percentage in G0/GI phase decreased significantly （P 〈 0. 001 ）, and the ability in angiogenesis was weakened. However, the ex- pression levels of P21, active easpase-3 as well as caspase-9 proenzyme and active caspase-9 were significantly higher than those in control group （P 〈 0. 001）. Conclusion SAHA inhibited the proliferation and angiogenesis in vitro of HUVECs and up-regulated the expressions of P21, caspase-3 and caspase-9, which provided a novel pathway for therapy of tumors.