Saikosaponin-d Inhibits Proliferation And Induces Poptosis In DU145 Human Prostate Cancer Cells

Prostate cancer is one of the most common diagnosed cancers among men. There is a large number of new cases and deaths every year,not only endanger people’s lives and health, but also bring the family and society heavy pressure and economic burden.With the developmentof diagnosis and therapy progress in recent years, the mortality rate of prostate cancer has been significantly reduced. Though prostate cancers are initially treatable, the androgen-insensitive or hormone-refractory recurrent cases of prostate cancer are not responsive to current therapies. There is an urgent need for novel therapeutic agents. China’s traditional Chinese medicine as a natural product,has thousands of years of clinical application, and has made significant achievements in anti-cancer therapy, characterized by low side effects and toxicity.Saikosaponins(SS) are triterpene saponins isolated from the root of Bupleurum falcatum L.(Umbelliferae). Because all SS have common steroid-like structure, they were expected and also confirmed to have some steroid-related pharmacological activities. SS has been widely used for its various pharmacological effects. So far, at least 8 types of SS have been identified, and among which saikosaponin d(SSd) is believed to be the most active component. Many studies have reported the pharmacological effects of SSd, including anti-inflammatory, regulate immune system,anti-tumor, treatment of renal disease, and antihepatic fibrosis properties, is a natural compound having important application value. In recent years, many Chinese medicine researchers conducted SSd in vivo tumor screening experiments and confirmed the inhibit effects of SSd in a variety of tumors. Recent studies have reported that SSd could potently inhibit the proliferation of many malignant cancer cells. However, effect of SSd on human prostate cancer cell lines remains under investigation. The aim of this study is to investigate the inhibitory activity of SSd on androgen-independent prostate cancer cell line DU145 and the mechanisms involved.DU145 cell morphology change will be observed by inverted phase contrast microscope. Effect of SSd on cell viability was measured using MTT method andKi67 immunocytochemistry staining. Hoechst 33258 staining was used to evaluate morphology change of nuclear. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. To further investigate the underling mechanism,intracellular reactive oxygen species(ROS), mitochondrial membrane potential(MMP) were evaluated by flow cytometry method. Protein expression of apoptosis-related proteins(Bcl-2, Bax, cytochrome C, Caspase-3 and p53) and cell cycle regulation protein(such as p21, cyclin D1, CDK4 and CDK6 protein) were detected by Western blot. At last, trypan blue was used to observe the cytotoxicity of SSd on mouse spleen cells. The results are as follows:1. SSd inbited the proliferation of DU145 cells in a concentration- dependent by MTT and Ki67 immunocytochemistry staining;2. SSd induced apoptosis in the DU145 human prostate cancer cells in a concentration and time-dependent manner by using Hoechst 3328 staining and flow cytometry;3. Flow cytometry analysis showed SSd treatment arrest the cell cycle in G1 phase,and in a concentration and time-dependent manner;4. Addition of wide Caspase inhibitors Z-VAD-FMK showed no significant change in the cell cycle, but showed a significantly inhibition of apoptosis;5. Flow cytometry analysis showed no significant change of SSd treatment on DU145 cells ROS levels;6. Flow cytometry analysis showed the decrease of DU145 cell mitochondrial membrane potential in a concentration-dependent manner;7. Western blot analysis revealed a significant increase in the protein expression of Bax, downregulation of Bcl2 protein, increased levels of cytoplasmic cytochrome C,and induced cleavage of Caspase-3 of DU145 cells after SSd treatment;8. Western blot analysis revealed the significant increase of p53 and p21 protein and reduced the expression level of Cyclin D1 and CDK4, CDK6 in SSd treated DU145 cells;9. SSd down-regulated expression of phosphorylated AKT protein, phosphorylated ERK protein, and down FOXO3 a protein phosphorylation levels in a concentration dependent manner.Thus, our present study got the following conclusions:1. We first showed that SSd can inhibit the proliferation of human prostatecarcinoma DU145 cells in a dose and time dependent manner, this inhibitory effect was achieved by inducing apoptosis and cell cycle arrest in G0 / G1 phase.2. SSd induced apoptosis of DU145 cells via the mitochondrial pathway, including up-regulating of pro-apoptotic protein Bax and inhibiting anti-apoptotic protein Bcl-2,promoting the dissipation of mitochondrial membrane potential, resulting in the release of Cyto c, activation of Caspase-3, then induced apoptosis.3. Cell cycle arrest in DU145 cells by SSd was associated with the up-regulated expression of p21, p53 protein and down regulation of Cyclin D1, CDK4 and CDK6 protein levels.4. Mitochondrial dysfunction induction is a major cause of cell proliferation inhibition of SSd in DU145 cells, and cell cycle arrest is a secondary cause.5. SSd can reduced the expression of phosphorylated ERK and AKT proteins, and inhibit the phosphorylation of FOXO3 a protein, which is a tumor suppressor and plays an important role as a transcription factor in regulating the cell cycle and apoptosis. So the results suggested that SSd may act by regulating the expression of FOXO3 a protein in prostate cancer cells.In conclusion, our results revealed that SSd can inhibit the proliferation of DU145 prostate cancer cells and its mechanisms involved to provide a theoretical basis for the development of an effective therapeutic agent in prostate cancer in future.