硫利达嗪诱导人卵巢癌细胞凋亡及其机制研究

Thioridazine induce apoptosis in human ovarian cancer cells by elevating reactive oxygen species

目的探讨多巴胺受体阻断剂硫利达嗪(thioridazine)对卵巢癌细胞株SKOV3、A2780增殖、凋亡的影响及其可能的作用机制。方法用不同浓度硫利达嗪(0、5、10、15、20μmol/L)作用SKOV3、A2780细胞,CCK8法检测细胞增殖,流式细胞仪及DAPI染色检测细胞凋亡,DCFH-DA法染色检测ROS水平,彗星实验观察细胞核DNA损伤情况,JC-1染色检测线粒体膜电位变化,Western blot检测P53、Bax、Bcl-2、细胞色素C、active Caspase-3蛋白的表达。结果硫利达嗪可抑制人卵巢癌细胞SKOV3、A2780的增殖,呈一定的剂量效应关系(P<0.05),浓度为15μmol/L时,可产生明显增殖抑制效应;流式细胞仪检测显示处理组(15μmol/L硫利达嗪作用24 h)凋亡率明显高于对照组(P<0.05);DAPI染色结果:处理组出现明显细胞核固缩、碎裂及凋亡小体;DCFH-DA染色处理组ROS水平升高(P<0.05);彗星实验结果提示处理组出现明显的DNA损伤;JC-1染色发现处理组线粒体膜电位较对照组下降(P<0.05);Western blot实验发现处理组P53、Bax、胞质细胞色素C、active Caspase-3表达上调,Bcl-2、线粒体细胞色素C表达下调。结论硫利达嗪可能通过诱导细胞内ROS升高损伤DNA,激活线粒体凋亡途径而诱导人卵巢癌SKOV3、A2780细胞凋亡。

Objective To determine the effect of thioridazine, a dopamine D2 receptor （DRD2） inhibitor, on the proliferation and apoptosis of ovarian cancer cell lines and investigate the potential mechanism. Methods After being treated with thioridazine （0, 5, 10, 15 and 20 μmol/L）, the proliferation of ovarian cancer cell lines SKOV3 and A2780 was respectively measured by CCK-8 assay, and cell apoptosis was determined by DAPI nuclear staining and flow cytometry. DCFH-DA staining was employed to observe the level of reactive oxygen species （ROS）. Comet assay was applied to detected nuclear DNA damage. JC-1 staining was used to detect mitoehondrial membrane potential. Tile expression of P53, Bax, Bel, cytochrome C and active Casepase-3 was assessed by Western blotting. Results The proliferation of SKOV3 and A2780 was suppressed by thioridazine in a dose-dependent manner （ P 〈 0.05 ）. Flow cytometry indicated that the apoptotic rate was significantly higher in the cells after thioridazine treatment （P 〈 0. 05 ） , and DAPI staining showed typical apoptotic features, including karyopyknosis, fragmentation, and apoptotic bodies. DCFH-DA staining showed the level of ROS was increased compared to control group （ P 〈 0. 05 ）. Comet assay suggested increased DNA damage, and JC-1 staining indicated decreased mitochondrial membrane potential compared to control group. Western blot analysis found that the expression of 1753, Bax, Cytoplasm cytoehrome C, active Caspase-3 was increased, while that of Bcl, mitochondrial cytochrome C was decreased. Conclusion Thioridazine may induces apoptosis via DNA damage and activation of mitochondrial pathway in ovarian cancer SKOV3 and A2780 cells