鸦胆子苦醇抑制人前列腺癌DU145细胞生长及作用机制

Inhibitory effects of brusatol on human prostate cancer cells DU145 and its molecular mechanism

关键词:  鸦胆子苦醇  人前列腺癌DU145细胞  凋亡  MAPK

Key words:  brusatol  human prostate cancer cells DU145  apoptosis  MAPK

摘要:

中药鸦胆子是一种常用的抗肿瘤中草药,鸦胆子苦醇是来源于鸦胆子的主要成分。该研究探讨了鸦胆子苦醇(brusatol)对人前列腺癌DU145细胞的生长抑制及其作用机制。采用四甲基偶氮唑盐(MTT)法检测鸦胆子苦醇对不同细胞株的生长抑制情况,以及不同浓度的鸦胆子苦醇对DU145细胞的增殖抑制率; 应用Hoechst 33258 染色法观察鸦胆子苦醇处理DU145细胞后所发生的形态学变化; 分别采用PI单染及Annexin-V-FITC 双染法流式细胞术分析细胞周期分布个凋亡率的变化; 以Western blot测定鸦胆子苦醇对MAPK信号通路相关蛋白表达的影响。结果表明:鸦胆子苦醇对人前列腺癌DU145细胞的抑制作用更为显著,并且可以时间和剂量依赖性地抑制人前列腺癌DU145细胞的生长,其半数有效抑制浓度IC₅₀为(0.27±0.04)μmol·L^-1; 鸦胆子处理DU145细胞后,Hoechst 33258染色可见到明显的凋亡特征; 细胞周期图中可见明显的亚二倍体峰,且随着作用时间的延长凋亡比例增加,FCM检测鸦胆子苦醇作用24 h后凋亡图中,可见凋亡的发生; Western blot检测表明鸦胆子苦醇处理后可使磷酸化的p38和JNK表达增加,使磷酸化的ERK表达降低。鸦胆子苦醇能显著抑制DU145细胞增殖,诱导DU145细胞凋亡。磷酸化的P38和JNK的表达增加,但磷酸化的ERK表达下降,这表明MAPK途径的活化可能是鸦胆子苦醇对DU145细胞生长抑制的作用机制之一。因此,鸦胆子苦醇是潜在的抗前列腺癌药物,有必要进一步在动物水平阐明其抗前列腺癌活性。

Abstract:

Fructus Bruceae is a Chinese Traditional Medicine that commonly used for the treatment of tumor diseases. Brusatol is one of the major active components in Fructus Bruceae. This study was to explore the inhibitory effects of brusatol against proliferation of human prostate cancer DU145 cells, and the molecular mechanism of apoptosis induced by brusatol was further investigated. The inhibitory activities of brusatol against human prostate cancer cells DU145 and PC3,hepatocellular carcinoma cell HepG2,human breast adenocarcinoma cell MCF-7,human colon adenocarcinoma cell HT-29,human pulmonary carcinoma cell A549 were assessed by MTT assay. The time-and concentration-dependent inhibition by brusatol on the most sensitive DU145 cells were further studied,and Hoechst 33258 staining was used to observe cellular morphologic changes. The distribution of cell cycle and apoptosis were analyzed by flow cytometry through PI and Annexin-V/FITC-PI double-labeled staining. To further analyze the possible mechanism of cell apoptosis,we investigated the protein expression levels of MAPK signaling pathway in DU145 cells after treatment with brusatol by Western blot. At the same concentration,brusatol showed the most potent inhibition on the proliferation of DU145 cells in the MTT assay. Furthermore,brusatol was found to inhibit DU145 cell growth in a time-and concentration-dependent manner. The IC₅₀ of the 48 h time course was(0.27±0.04)μmol·L^-1. Apoptosis was measured by Hoechst 33258 staining,which showed increased fragmented chromatin and apoptotic bodies after the treatment with 0.25 μmol·L^-1 of brusatol as compared with the solvent control. A typical subdiploid peak was observed by flow cytometry,and the ratio of subdiploid peak was further increased with the time. Apoptosis of DU145 cells was analyzed by AnnexinV/FITC-PI staining and flow cytometry detection. The apoptosis rate was increased from 0.7% to 10.6% after the treatment of brusatol for 24 h,which confirmed that brusatol could induce apoptosis. Western blot analysis showed that brusatol can affect the expression levels of MAPK superfamily at a concentration of 0.25 μmol·L^-1 after incubation for 45 min,1.5,3,6,12 and 24 h. Brusatol selectively increased the phosphorylation of p38 and JNK,while decreased the phosphorylation of ERK1/2,all in time-dependent manners. Brusatol could significantly inhibit the proliferation of DU145 cells at a dose-and time-dependent manner,and it could also induce cell apoptosis. The increased phosphorylation of p38 and JNK,while decreased phosphorylation of ERK1/2 suggested that mitogen-activated protein kinase(MAPK)pathway might be involved in the brusatol-induced apoptosis on DU145 cells. Thus brusatol is a potential anticancer drug against the prostate cancer. Further studies to reveal its anticancer properties at the animal level are warranted.