CAJ | 학술논문

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.
중약아담자시일충상용적항종류중초약,아담자고순시래원우아담자적주요성분.해연구탐토료아담자고순(brusatol)대인전렬선암 DU145세포적생장억제급기작용궤제.채용사갑기우담서염(MTT)법검측아담자고순대불동세포주적생장억제정황,이급불동농도적아담자고순대 DU145세포적증식억제솔；응용Hoechst 33258염색법관찰아담자고순처리 DU145세포후소발생적형태학변화；분별채용 PI 단염급Annexin-V-FITC 쌍염법류식세포술분석세포주기분포개조망솔적변화；이 Western blot 측정아담자고순대MAPK 신호통로상관단백표체적영향.결과표명：아담자고순대인전렬선암 DU145세포적억제작용경위현저,병차가이시간화제량의뢰성지억제인전렬선암 DU145세포적생장,기반수유효억제농도 IC50위(0．27±0．04)μmol·L-1；아담자처리 DU145세포후,Hoechst 33258염색가견도명현적조망특정；세포주기도중가견명현적아이배체봉,차수착작용시간적연장조망비례증가,FCM 검측아담자고순작용24 h 후조망도중,가견조망적발생；Western blot 검측표명아담자고순처리후가사린산화적 p38화 JNK 표체증가,사린산화적 ERK표체강저.아담자고순능현저억제 DU145세포증식,유도 DU145세포조망.린산화적 P38화 JNK 적표체증가,단린산화적 ERK 표체하강,저표명 MAPK 도경적활화가능시아담자고순대 DU145세포생장억제적작용궤제지일.인차,아담자고순시잠재적항전렬선암약물,유필요진일보재동물수평천명기항전렬선암활성.
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 apop-tosis 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 colonadenocarcinoma cell HT-29,human pulmonary carcinoma cell A549 were assessed by MTT assay.The time-and con-centration-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 an-alyzed 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 po-tent 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 ap-optosis rate was increased from 0.7% to 10.6% after the treatment of brusatol for 24 h,which confirmed that brusa-tol could induce apoptosis.Western blot analysis showed that brusatol can affect the expression levels of MAPK su-perfamily 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 phosphoryla-tion 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.