麦冬皂苷D’通过RIP1/MLKL诱导前列腺癌PC3细胞程序性坏死

Ophiopogonin D' induces necroptosis in prostate cancer PC3 cells through RIP1/MLKL pathway

目的研究天然皂苷类化合物麦冬皂苷D’(Ophiopogonin D’,OPD’)对雄激素非依赖的前列腺癌PC3细胞的抗肿瘤效应及其机制。方法不同浓度的OPD’处理前列腺癌PC3细胞后,MTT实验检测细胞生长能力,流式细胞术检测Annexin V-Fitc和PI双染,Western blot检测程序性坏死相关蛋白——受体相互作用蛋白1(receptor interacting protein-1,RIP1)、混合谱系激酶结构区域样蛋白(mixed lineage kinase domain-like,MLKL)的表达,结晶紫染色、透射电镜观察细胞形态学的改变。结果OPD’抑制PC3细胞生长活性,24 h IC50值为(6.25±0.31)μmol/L。5μmol/L OPD’处理24 h诱导PC3细胞凋亡和坏死,5μmol/L OPD’处理6 h PC3细胞在晚期凋亡/坏死象限呈明显的时间依赖关系(r=0.728,P<0.001)。OPD’诱导PC3细胞Caspase非依赖性死亡,且其死亡能被程序性坏死(necroptosis)特异性抑制剂necrostatin-1(Nec-1)所抑制(P=0.047),表明OPD’诱导PC3细胞程序性坏死。OPD’下调Cleaved-RIP1、上调RIP1的表达;上调p-MLKL四聚体的表达,并呈明显的时间依赖关系(r=0.907,P=0.005)。形态学上OPD’诱导PC3细胞坏死样改变。结论 OPD’抑制前列腺癌细胞生长具有明显的抗肿瘤效应,而诱导其通过RIP1/MLKL通路发生程序性坏死可能是其发挥抗肿瘤效应的机制之一。

Objective To determine the anti-tumor effect of Ophiopogonin D'( OPD'),a natural saponin compound,against androgen-independent prostate cancer PC3 cells,and investigate the underlying mechanism. Methods PC3 cells were treated with different concentrations of OPD '( 0,1,2. 5,5 and10 μmol/L). MTT assay,flow cytometry,Western blotting,crystal violet staining,and transmission electron microscopy were used to investigate the effects of OPD'on cell viability,Caspase-independent cell death,necroptosis,expression levels of necroptosis related proteins,receptor interacting protein-1( RIP1) and mixed lineage kinase domain-like protein( MLKL),and morphological changes,respectively. Results OPD 'treatment significantly inhibited the growth in PC3 cells,with 24-hour IC50 of 6. 25 ± 0. 31 μmol/L. OPD'treatment of 5 μmol/L for 24 h could induce necrosis in PC3 cells. Meanwhile,OPD'treatment of 5 μmol/L for 6 h induced late apoptosis/necrosis in a time-dependent manner( r = 0. 728,P < 0. 001). OPD'also induced Caspase-independent cell death in PC3 cells,which could be inhibited by necrostatin-1( Nec-1),a specific inhibitor of necroptosis( P = 0. 047). This indicated that OPD'induced necroptosis in PC3 cells.OPD'reduced the expression levels of Cleaved-RIP1,while increased the levels of RIP1 and p-MLKL in PC3 cells,and the expression of p-MLKL was elevated in a time-dependent manner( r = 0. 907,P = 0. 005).Morphological observation displayed that OPD'induced necrosis-like changes in PC3 cells. Conclusion OPD' exerts obvious anti-cancer effects by inhibiting cell viability in prostate cancer cells,and through activating RIP1/MLKL pathway to induce necroptosis might be one of underlying mechanisms.