辛二酰苯胺异羟肟酸对人卵巢癌紫杉醇耐药细胞OC3/P存活和凋亡的影响

Effect of suberoylanilide hydroxamic acid on survival and apoptosis of human paclitaxel-resistant ovarian cancer OC3 / P cells

目的 研究辛二酞苯胺异羟肟酸（SAHA）对人卵巢癌紫杉醇耐药细胞OC3/P存活和凋亡的影响。方法 SAHA 4~64 μmol·L-1与OC3/P细胞作用6，12，24或48 h，用倒置显微镜观察细胞形态的变化，瑞氏吉姆萨染色观察细胞质和细胞核形态的变化，MTT法检测细胞存活率，AnnexinⅤ-FITC/PI双染流式细胞术测定细胞凋亡率，用透射电镜观察细胞内超微结构的变化。结果 倒置显微镜下可见，SAHA 4，16和64 μmol·L-1与OC3/P细胞孵育24 h，可引起OC3/P细胞皱缩，细胞贴壁不良，折光率减弱，活细胞数目减少，且随着SAHA浓度升高细胞形态改变更明显。瑞氏吉姆萨染色发现，SAHA 64 μmol·L-1与OC3/P细胞孵育24 h，使细胞体积减小、细胞质凝集和核固缩。MTT法检测结果表明，SAHA 4~64 μmol·L-1对OC3/P细胞存活具有抑制作用，并存在明显的时间和浓度效应关系，其中SAHA 64 μmol·L-1作用 6，12，24和48 h对OC3/P细胞存活的抑制率分别为（7.1±1.9）%，（14.6±2.0）%，（36.8±2.7）%和（83.3±3.0）%（r=0.891，P〈0.05），SAHA 4，8，16，32和64 μmol·L-1作用48 h对OC3/P细胞存活的抑制率分别为（28.8±1.2）%，（34.8±4.3）%，（52.3±2.8）%，（61.3±4.9）%和（83.3±3.0）%（r=0.966，P〈0.05）。流式细胞术检测结果表明，SAHA 4，16和64 μmol·L-1作用48 h均可诱导OC3/P细胞凋亡（P〈0.05，P〈0.01）。透射电镜观察结果表明，SAHA 4，16和64 μmol·L-1作用24 h，OC3/P细胞核出现典型的凋亡形态变化。结论 SAHA可抑制人卵巢癌紫杉醇耐药细胞OC3/P的存活并诱导其凋亡。

OBJECTIVE To study the effect of suberoylanilide hydroxamic acid （SAHA） on survival and apoptosis of human paclitaxel-resistant ovarian cancer OC3/P cells. METHODS OC3/P cells were treated by SAHA with the concentrations of 4, 8, 16, 32 and 64 μmol ·L-1 for 6, 12, 24 or 48 h. Morphological changes of OC3/P cells were observed under inverted microscope, and the changes of cell nucleus were assessed by Giemsa-Wright staining. The viability of OC3/P cells was detected with MTT assay. The apoptosis rates of OC3/P cells were analyzed by AnnexinⅤ-FITC/PI double staining assay with flow cytometry. Transmission electron microscopy was used for assessing the cells ultrastructure. RESULTS After incubation with SAHA 4,16 and 64 μmol·L-1 for 24 h, OC3/P cells became shrinkage, poor adherence, less refractive, which were more obvious with the increase in SAHA concentration. Giemsa-Wright staining showed that OC3/P cells became cytoplasmic agglutination and karyopyknosis after treated with SAHA 64 μmol·L-1 for 24 h. MTT assay revealed that SAHA could inhibit OC3/P cell survival in a concentration-dependent manner （P〈0.05） and time-dependent manner （P〈0.05）. The inhibitory ratios of SAHA at concentration 64 μmol·L-1 for 6, 12, 24 and 48 h were（7.1±1.9）%,（14.6±2.0）%,（36.8±2.7）% and （83.3±3.0）%, respectively（r=0.891,P〈0.05）, and SAHA 4-64 μmol·L-1 for 48 h were（28.8±1.2）%,（34.8±4.3）%,（52.3±2.8）%,（61.3±4.9）% and （83.3±3.0）%, respectively（r=0.966,P〈0.05）. The results of flow cytometry assay revealed that SAHA 4, 16 and 64 μmol·L-1 for 48 h could induce apoptosis of OC3/P cells （P〈0.05, P〈0.01）. The apoptosis characteristics in OC3/P cells were also confirmed by transmission electron microscopy. CONCLUSION SAHA can inhibit the survival and induce apoptosis of human paclitaxel-resistant ovarian cancer OC3/P cells.