PsL5F诱导人未分化甲状腺癌FRO细胞凋亡的机制研究

Molecular Mechanism of Apoptosis Induced by PsL5F in Human Anaplastic Thyroid Carcinoma FRO Cells

目的研究半边旗5F(PsL5F)对人未分化甲状腺癌FRO细胞的凋亡诱导及其分子机制。方法用PsL5F处理FRO细胞,MTT法测定其对FRO细胞的生长抑制作用;Annexin V-FITC/PI标记法与流式细胞术联用检测PsL5F对FRO细胞的凋亡诱导;用CM-H2DCFDA和Di OD6(3)标记在流式细胞仪上分析PsL5F对FRO细胞内活性氧(ROS)水平和线粒体膜电位(MMP)的影响;Western blot方法分析Bax、Cyto C、凋亡诱导因子(AIF)及截断PARP的水平变化;Caspase-3活性用Caspase-3比色分析试剂盒测定。结果PsL5F对FRO细胞具有显著的生长抑制作用,且呈现剂量和时间依赖性;PsL5F可诱导FRO细胞凋亡,表现出在100 mg/L浓度下,磷脂酰丝氨酸(PS)外翻细胞的比例呈时间依赖性逐渐增加;用100 mg/L PsL5F处理FRO细胞,在1 h时细胞内ROS水平即明显升高,ROS抑制剂还原型谷胱甘肽(GSH)可抑制PsL5F诱导的细胞内ROS水平升高和细胞凋亡;100mg/L PsL5F处理可使FRO细胞MMP逐渐降低,GSH可抑制PsL5F诱导的MMP降低;同时,线粒体膜蛋白组分中Bax和细胞液蛋白组分中Cyto C、AIF逐渐增加;PsL5F处理使FRO细胞Caspase-3活性逐渐升高及其作用底物PARP断裂。结论PsL5F对人未分化甲状腺癌FRO细胞的生长抑制作用是通过诱导细胞凋亡进行的。其中,细胞内ROS水平升高起到了非常重要的第二信使作用,线粒体是其作用的重要靶点。细胞凋亡是通过Bax转位到线粒体膜、引起MMP降低、Cyto C和AIF释放到细胞液、激活Caspases级联反应而进行的。

Objective To investigate the apoptosis induced by Pteris semipnnata L 5F（PsL5F） in human anaplastic thyroid carcinoma FRO cells and its molecular mechanism.Methods Human anaplastic thyroid carcinoma FRO cells were treated with PsL5F,and the growth inhibition rate was evaluated by MTT assay.The cell apoptosis rate was assessed by Annexin V-FITC fluorescence staining and flow cytometry.Intracellular reactive oxygen species（ROS） levels were analyzed by CM-H2DCFDA fluorescence staining and flow cytometry.Mitochondrial membrane potential（MMP） was measured by DiOD6（3） fluorescence staining and flow cytometry.The levels of Bax,Cyto C,AIF and cleaved PARP were analyzed by Western blotting.The activity of caspase-3 was assayed by caspase-3 colorimetric assay kit.Results PsL5F has significant growth inhibitory action on FRO cells in dose and time dependent manners.Under the treatment of 100 mg/L of PsL5F,the percentage of apoptotic cells with phosphatidylserine（PS） externalization was gradually increased in time dependent manner.The rise of ROS level in FRO cells was observed as early as 1h after treated with PsL5F.The elevation of intracellular ROS levels and cell apoptosis could be inhibited by glutathione（GSH）,a scavenger of ROS.The MMP in FRO cells was gradually reduced by PsL5F,and the reduction of MMP can be inhibited by GSH.Meanwhile,the levels of Bax in fraction of mitochondrial membrane,Cyto C and AIF in fraction of cytosol were gradually increased.PsL5F can cause the increase of caspase-3 activity and cleavage of PARP,a substrate of caspase-3. Conclusion PsL5F can inhibit growth of human anaplastic thyroid carcinoma FRO cells through inducing apoptosis.The rise of ROS levels in FRO cells plays important role as a secondary messenger in apoptosis induced by PsL5F.Mitochondrium is an important target of PsL5F.Cell apoptosis induced by PsL5F in FRO cells was carried out through translocation of Bax to mitochondrial membrane,reduction of MMP,release of Cyto C and AIF from mitochondria to cytosol,and activation of caspases cascade reaction.