辛二酰苯胺异羟肟酸诱导人卵巢癌细胞OC3自噬作用

Effect of suberoylanilide hydroxamic acid on autophagy of human ovarian cancer OC3 cells

目的探讨辛二酰苯胺异羟肟酸(SAHA)是否诱导人卵巢癌细胞OC3发生自噬。方法 SAHA0.05,0.25,1.25,6.25和31.25μmol·L-1处理OC3细胞24 h后,倒置显微镜吉姆萨和-瑞氏染色观察细胞形态变化;四甲基偶氮唑蓝(MTT)法检测细胞存活;流式细胞术检测自噬微管相关蛋白轻链3B(LC3B)的表达;AO/EB双染色观察红色酸性自噬囊泡的出现;电镜直接观察自噬囊泡的结构。结果不同浓度SAHA处理后,OC3细胞形态呈不规则梭形或多角形,细胞空泡化增多,折光性差。MTT结果表明,不同浓度SAHA处理对OC3细胞的存活具有明显的抑制作用,并存在时间和浓度效应关系,浓度相关系数分别为r12 h=0.898,r24 h=0.976和r48 h=0.952(P<0.05),SAHA 0.05,0.25,1.25,6.25和31.25μmol·L-1时,时间相关系数分别为0.999,0.654,0.999,0.869和0.922(P<0.05)。SAHA与OC3细胞作用24 h后,AO/EB双染色可见红色酸性自噬囊泡出现;进一步电镜观察,可观察到自噬囊泡结构;流式细胞术检测结果表明,与正常对照组相比,SAHA 0.05,0.25,1.25,6.25和31.25μmol·L-1处理OC3细胞24 h后,LC3B阳性细胞比率显著升高,分别为(19.4±2.4)%,(28.5±3.4)%,(34.6±3.9)%,(38.6±3.2)%和(61.8±1.0)%(P<0.05)。结论SAHA可能通过诱导人卵巢癌细胞OC3发生自噬而杀伤肿瘤细胞。

OBJECTIVE To evaluate the effect of suberoylanilide hydroxamic acid （SAHA） on autophagy of human ovarian cancer OC3 cells. METHODS OC3 cells were treated with SAHA 0.05, 0.25, 1.25,6.25 and 31.215 μmol · L^-1 for 24 h, and then stained by Giemsa-Wright＇s. The morphological changes of OC3 cells were observed under an inverted microscope and cell proliferation was detected by MTT assay. The autophagy related proteins were analyzed by flow cytometry. The cell ultrastructure changes were identified by transmission electron microscopy and autophagic vacuoles of OC3 cells were observed by AO/EB double staining. RESULTS After treatment with SAHA 0.05-31.25 μmol· L^-1, the morphology of OC3 cells became irregularly spindle-shaped with more vacuolization and less refraction. The activity and proliferation of OC3 cells were significantly decreased in a time-dependent and SAHA concentration dependent manner by MTT assay. Concentration-dependent correlation coeffi- cients were r,2 h=0.898, r24 h=0.976 and r48 h=0.952 （P〈0.05）, respectively. SAHA time correlation coeffi- cients were 0.999,0.654,0.999,0.869 and 0.922 （P〈0.05）, respectively. Using AO/EB double staining, the amount of acidic autophagic vacuoles was augmented. With transmission electron microscopy, the structure of autophagic vacuoles could be seen clearly. Flow cytometry results showed that the positive rate of LC3B cells was significantly increased in SAHA 0.05-31.25 μmol·L^-1, which was （19.4±2.4）%, （28.5±3.4）%, （34.6±3.9）%, （38.6±3.2）%, and （61.8±1.0）%, respectively. CONCLUSION SAHA can inhibit and kill human ovarian cancer cells OC3 by inducing autophagy.