Studies On The Inhibitory Effects Of The Constituents From Brucea Javanica And Helleborus Thibetanus On Prostate Cancer Cells

Prostate cancer is the second most common diagnosed cancer after lung cancer worldwide,and it is the third most common cause of cancer deaths in developed countries in man.Despite recent progress in diagnostic and multimodal therapies of initial prostate cancer, there is no effective therapy in treating androgen-independent cases. The mortality rate associated with recurrent prostate cancer is still very high. So, there is great demand for effective novel therapeutic agents. It is a hot field for the research and development of the new anticancer drugs with strong antitumor activity but low side effects from Chinese herbal medicines or natural medicines.Purpose: The thesis includes two parts. In part 1, we aimed to investigate the inhibitory activity of brusatol(YD-1), bruceine A(YD-2) and bruceantiol(YD-3) on the proliferation of androgen-independent prostate cancer cell line DU145 and the molecular mechanisms involved so as to provide a scientific basis for the treatment of prostate cancer with this kind of compounds. In part 2, the objective is to assess the inhibitory activities of chemical institutents of Helleborus Thibetanus Franch on androgen-independent prostate cancer cell line, and discuss the structure-activity relationship.Methods: The cell viability activity of the chemical constituents of two Chinese herb medicines on different tumor cells and prostate cancer cells was evaluated by MTT assay.Hoechst 33258 staining and DNA fragmentation analysis experiment was used to examine the apoptotic induction activity of compounds on prostate cancer DU145 cell. Cell cycle distribution, cell apoptosis, intracellular ROS, mitochondrial membrane potential were evaluated by flow cytometry method. The expression levels of Bax, Bc I-2, caspase-3, PARP and MAPK proteins were measured by western blot.Results: MTT results showed that YD 1—3 dose-dependently and time-dependently decreased the viability of DU145 cells. The IC50 values of them were 0.37±0.03 μM,0.60±0.19 μM and 0.41±0.03 μM, respectively. These compounds could induce apoptosis in DU145 cells as observed by optical microscope observation, DNA fragmentation analysis andFCM. Hoechst 33258 results showed that these compounds caused significant nuclear condensation in DU145 cells in a dose-dependent manner(0.0625, 0.125, 0.25, 0.5 μM). The DNA ladder analysis showed YD-1 caused DNA fragmentation. Flow cytometry analysis found that YD 1—2 significantly arrested cell cycle in S phase, while YD-3 arrested cells in G2/M phase. The compound induced apoptosis of DU145 cells by using Annexin V/FITC-PI double staining and flow cytometry detection. The results of FCM indicated that the apoptosis rate was(10.6±1.1)%,(6.9±0.8)%,(10.6±0.9)% respectively by the treatment of YD-1(0.25μM), YD-2(0.5 μM)and YD-3(0.3 μM) for 24 h. The ROS level in cancer cells increased after treatment by these compounds. The decrease of mitochondrial membrane potential suggested that these compounds may induce apoptosis through mitochondrial pathway. The expression levels of cytochrome c, Bax, Bcl-2 and caspase-3 were measured by western blotting. Western blot analysis demonstrated that YD-1 induced apoptosis in DU145 cells is associated with disruption of mitochondrial membrane potential, down-regulation of Bcl-2,and up-regulation of Bax, and activation of caspase-3 and PARP. Moreover, these compounds selectively altered the phosphorylation state of members in the MAPK superfamily, decreased phosphorylation of ERK and activated phosphorylation of p38 in a time-dependent manner.The 19 compounds in Helleborus Thibetanus Franch showed different degree of inhibitory activities on prostate cancer DU145 and PC3 cells.Conclusion:1. YD 1—3 could cause obvious growth inhibition of du145 cells in a dose- and time-dependent manner at the tested range of concentrations; Compounds suppression DU145 cells growth by inhibiting proliferation and inducing apoptosis; The mechanism of YD-1induced apoptosis might be down- regulation of Bcl-2, and up-regulation of Bax, and activation of caspase-3 and PARP. YD-2could induce prostate cancer cells DU145 apoptosis through P38 and ERK pathway. YD-3 could induce prostate cancer cells DU145 apoptosis through P38, JNK and ERK pathway. ERK pathway may be involved in the anti-proliferative mechanism, while the p38 pathway might participate in the induction of apoptosis caused by compounds on DU145 cells.2. Among the inhibitory activities of 19 compounds from Helleborus Thibetanus Franch,the bufadienolides showed the most potent inhibitory activities against prostate cancer cell and the bufadienolide glycosides showed stronger inhibitory activities than the bufadienolide aglycones. Bufadienolides with A/B cis configuration showed stronger inhibitory activities than those with A/B trans configuration. The ordinary steroidal compounds showed only mild or very weak inhibitory activities.