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

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 Hep G2,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-1of 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 Annexin V / 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-1after 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 doseand 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.