桔梗皂苷D通过FOXO3a通路介导前列腺癌PC-3细胞程序性坏死  

Platycodin D induces necroptosis of prostate cancer PC-3 cells through FOXO3a pathway

作　　者：宋伟[1,2] 王佳佳 王贺[1] 朱明星[1] 吴长蓬 贺英 黎娜[1] 卢宗亮 许红霞[1] SONG Wei;WANG Jiajia;WANG He;ZHU Mingxing;WU Changpengt;HE Ying;LI Na;LU Zongliang;XU Hongxia(Department of Nutrition, Institute of Field Surgery, Daping Hospital, Army Medical University, Chongqing 400042, China;No. 92143 Force, Chinese People＇s Liberation Army, Sanya 572021, Hainan Province, China;Department of Clinical Nutrition, Chongqing People＇s Hospital, Chongqing 400014, China)

机构地区：[1]陆军军医大学大坪医院野战外科研究所营养科 [2]解放军92143部队 [3]重庆市人民医院临床营养科

出　　处：《肿瘤》2018年第2期85-93,共9页Tumor

基　　金：国家自然科学基金青年科学基金资助项目(编号:81603347)~~

摘　　要：目的:研究天然化合物桔梗皂苷D(platycodin D)对前列腺癌PC-3细胞程序性坏死的影响,并初步探索转录因子叉头转录因子O3a(forkhead transcription factor O3a,FOXO3a)在桔梗皂苷D诱导的PC-3细胞程序性坏死中的相关作用机制。方法:分别采用广谱的caspase抑制剂Z-VAD-FMK和特异性针对caspase-3的抑制剂AC-DEVD-CHO预处理PC-3细胞,再用不同浓度的桔梗皂苷D处理72 h,MTT法检测PC-3细胞的存活率。分别采用锥虫蓝染色及乳酸脱氢酶(lactic dehydrogenase,LDH)释放实验检测桔梗皂苷D对PC-3细胞膜完整性(锥虫蓝染色率和LDH释放率)的影响。蛋白质印迹法检测桔梗皂苷D对PC-3细胞中程序性坏死关键因子混合谱系激酶结构区域样蛋白(mixed lineage kinase domain-like,MLKL)、磷酸化MLKL(phospho-MLKL,p-MLKL)以及p-MLKL四聚体表达水平的影响,以及对FOXO3a及其下游分子自杀相关因子配体(factor-associated suicide ligand,FasL)和肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factorrelated apoptosis-inducing ligand,TRAIL)蛋白表达的影响。免疫荧光法检测FOXO3a蛋白在细胞核内的表达情况。采用脂质体转染法将FOXO3asiRNA和阴性对照(negative control,NC)-siRNA转入PC-3细胞,蛋白质印迹法检测siRNA对FOXO3a蛋白表达水平的影响。FOXO 3a基因沉默后,分别采用MTT法、锥虫蓝染色及LDH释放实验检测沉默FOXO 3a基因表达后桔梗皂苷D对PC-3细胞存活率和细胞膜完整性(锥虫蓝染色率和LDH释放率)的影响。结果:随着桔梗皂苷D浓度的提高,PC-3细胞的存活率明显降低,呈浓度依赖性;同一桔梗皂苷D浓度下,Z-VAD-FMK和AC-DEVD-CHO组PC-3细胞的存活率与对照组相比,差异均无统计学意义(P值均>0.05),提示桔梗皂苷D对PC-3细胞增殖的抑制作用是非caspase依赖性的。桔梗皂苷D处理PC-3细胞后,锥虫蓝染色率和LDH释放率均明显升高(P值均<0.05)。桔梗皂苷D可上调PC-3细胞中程序性坏死关键因子MLKL、p-MLKObjective： To investigate the effect of platycodin D on necroptosis of prostate cancer PC-3 cells, and to explored its mechanism related to forkhead transcription factor O3a （FOXO3a）. Methods： PC-3 cells were treated with caspase inhibitor Z-VAD-FMK or caspase-3-specific inhibitor AC-DEVD-CHO, and then were exposed to different concentrations of platycodin D for 72 h. The survival rate of PC-3 cells was detected by MTT method. The cell morphology and membrane integrity of PC-3 cells treated with platycodin D were detected by typan blue staining and lactic dehydrogenase （LDH） release test, respectively. The expression levels of key necroptosis factors including mixed lineage kinase domain-like （MLKL）, phospho- MLKL （p-MLKL） and its tetramer, as well as FOXO3a and its downstream molecules including factor-associated suicide ligand （FasL） and tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） proteins in PC-3 cells treated with different concentrations of platycodin D were detected by Western blotting. The expression level of FOXO3a protein in the nucleus of PC-3 cells was detected by immunofluorescence. After PC-3 cells were transfected with FOXO3a-siRNA or the negative control （NC）-siRNA by liposome, and the expression levels of FOXO3a, FasL and TRAIL proteins were detected by Western blotting. The cell viability and cell membrane integrity of PC-3 cells after FOXO3a gene silencing and treatment with platycodin D were detected by MTT method, typan blue staining and LDH release test, respectively. Results： The survival rate of PC-3 cells treated with platycodin D was decreased in a concentration-dependent manner. There was no significant difference in the survival rate of PC-3 cells treated with DMSO, Z-VAD-FMK and AC-DEVD-CHO combined with the same concentration of platycodin D （all P 〉 0.05）. After the treatment with platycodin D, the trypan blue staining rate and LDH release rate of PC-3 cells were significantly increased （both P 〈 0.05）. The expression

关 键 词：前列腺肿瘤 桔梗皂苷D 程序性坏死 叉头转录因子O3a 

分 类 号：R737.9[医药卫生—肿瘤]