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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 study the antitumor effect of natural saponin Ophiopogonin D ’(OPD’) on androgen-independent prostate cancer PC3 cells and its mechanism. Methods PCD cells were treated with different concentrations of OPD ’. MTT assay was used to detect the cell growth. Flow cytometry was used to detect Annexin V-Fitc and PI double staining. Western blot was used to detect the expression of apoptosis-associated protein-receptor interacting protein 1 (RIP1), mixed lineage kinase domain-like (MLKL) expression, crystal violet staining and transmission electron microscopy. Results OPD ’inhibited the growth of PC3 cells at 24 h IC50 of (6.25 ± 0.31) μmol / L. Apoptosis and necrosis of PC3 cells were induced by 5μmol / L OPD ’for 24 h, and the apoptosis / necrosis of PC3 cells treated by 5μmol / L OPD’ for 6 h showed a significant time dependence (r = 0.728, P <0.001). OPD ’induced Caspase-independent death in PC3 cells and its death was inhibited by necrostatin-1 (Nec-1) (P = 0.047), indicating that OPD’ induced PC3 cell programmedness Necrosis. OPD ’down-regulated Cleaved-RIP1, up-regulated the expression of RIP1; up-regulated the expression of p-MLKL tetramers in a time-dependent manner (r = 0.907, P = 0.005). Morphologically OPD ’induces PC3 cell necrosis-like changes. Conclusion OPD inhibits the growth of prostate cancer cells and has obvious antitumor effects. However, it may be one of the mechanisms by which OPD can induce apoptosis through RIP1 / MLKL pathway.