Saponin 6 Derived From Anemone Taipaiensis Induces Human Malignant Glioblastoma U87 Cell Apoptosis Via Regulation Of Fas And Bcl2 Family Proteins

Glioblastoma multiforme(GBM) is the most common form of intracranial malignant tumors among adults, which originates from the interstitial cells in the out layer of nerves, accounting for 70% in primary malignant brain tumors. Despite of proper treatment with radiotherapy and chemotherapy after operation, the rate of tumor recurrence stays high, which is due to the characteristics of invasiveness, fast growth and non-typical early clinical manifestation. The patients have a poor prognosis. The median survival is about 14.6 months and 3 year survival rate is only 10%. At present, the main treatment of drugs includes cisplatin, paclitaxel and carmustine(BCNU), etc. Because of the existence of drug resistant protein and abnormal DNA repair in tumor, the therapeutic response to anticancer drugs turn out to be unsatisfactory.Taking the blood-brain barrier, the blood-tumor barrier and the cell membrane permeability into consideration, the new drug research and development are imminent needs. A large number of studies has shown that, different from the traditional chemotherapy drugs, Chinese medicine extracted from natural plants can selectively kill cancer cells with little effect on normal cells. In addtion, the mechanism often includes a variety of ways such as apoptosis, autophagy and cell cycle control, etc. Therefore, it has potential development prospect.Saponin 6 of anemone taipaiensis(saponin6) derives from Anemone L. Previous studies have shown that some of the extract from anemone taipaiensis can inhibit the glioma proliferation. Different from previous studies of the extract, the monomer molecular structure of saponin 6 is mainly oleanolic acid, with C-3 into Oxygen glycosidies,C-28 into Free carboxyl and less C-3 oligosaccharide chain number, which can be inferred as high biological activity. Therefore, the research takes saponin 6 as researching subject, observing the effects of drug induced apoptosis in U87 malignant glioblastoma(U87 MG) cells and exploring the possible molecular mechanism of the drug. The experiment was divided into three parts:Part I: The effect of anemone taipaiensis saponin 6 on the proliferation of U87 MG cellsObjective: To compare the different concentrations of saponin 6’s effect on the proliferation of U87 MG cells. At the same time, to observe the effect of saponin6 on normal mouse hippocampus neuronal HT-22 cells.Methods: 1.Treating U87 MG cells with different concentrations(0.00、1.60、3.20、6.40、12.80 μg/m L)of saponin 6 for 24 hours and 48 hours. Comparing the different concentrations and reaction times of saponin 6’s effect on normal mice hippocampal neuronal HT-22 cells. Using CCK8 method to detect the effect of saponin 6 on the cell growth activity. 2. The effect of saponin 6 on cell cycle by PI staining flow cytometry analysis. 3. Using the SPSS20.0 and Graph Pad Prism 5.01 statistical software for statistical data analysis.Results: 1. Compared with normal culture conditions of U87 MG cells, saponin 6 significantly inhibited the viability of U87 MG cells, which was in a time- and dose- dependent manner. When the drug’s concentration increased to 2.53 μg/m L, 50% of U87 MG cells’ cell viability was inhibited. Saponin 6 had no obvious effect on the viability of HT-22 cells in low concentrations(<3.20 μg/m L). When the drug concentration came to 5.59 μg/m L, the viability inhibition rate of HT-22 cells reached 50%. 2. The cell cycle analysis showed that treatment groups decreased in S phase and blocked in G0/G1 phase with the inhibition of cell proliferation.Conclusion: 1. Saponin 6 optionally inhibited the viability of U87 MG cells in a time- and dosedependent manner. Under the concentration of 3.2 μg/m L, saponin 6 had little effects on HT-22 cells. 2. Saponin 6 could inhibit cell proliferation by affecting U87 MG cell cycle by blocking cell cycle in G0/G1 phase.Part Ⅱ: Saponin 6 of anemone taipaiensis induces the apoptosis in U87 MG cellsObjective: Analyzing saponin 6’s effect on the apoptosis in U87 MG cells;Methods: Treating U87 MG cells with different concentrations(2.53, 5.06 μg/m L)of saponin 6 for 24 hours. Cell apoptosis was assessed by flow cytometry using annexin V-FITC/propidium iodide(PI) double staining. DNA fragmentation and changes in nuclear morphology were examined by terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) staining and transmission electron microscopy(TEM), respectively.Results: TUNEL staining showed that, compared with the control group, saponin 6 could induce apoptosis in U87 MG cells in a dose-dependent manner. Annexin V-FITC / PI double staining flow cytometry disclosed that saponin 6 triggered early apoptosis in U87 MG cells. In treatment groups, Hoechst33342 staining revealed that saponin6 could cause typically apoptosis morphological changes including chromatin fragmentation and apoptotic bodies. Compared with the control group, transmission electron microscope was used to observe the obvious changes in apoptosis.Conclusion: Saponin 6 could induce apoptosis in U87 MG cells, which was associated with concentrations of the drug.Part Ⅲ: The mechanism of apoptosis in U87 MG cells induced by Anemone taipaiensis saponin 6Objective: to understand the mechanism of apoptosis in U87 MG cells induced by saponin 6Methods: Treating U87 MG cells with different concentrations(2.53, 5.06 μg/m L)of saponin 6 for 24 hours. Through the detection method of western blot analysis, observing the expression level of extrinsic apoptotic pathway related protein(Fas/Fas-L/caspase8), the intrinsic apoptotic pathway related protein(Bax/Bcl2/caspase9) and the executive apoptosis protein caspase3.Results: 1. Compared with the control group, after adding saponin 6, Western blot detected the expression of cleaved-caspase3 in treatment groups, which was in a significant dose-dependent manner. 2. At the same time, the protein expression of Fas, Fas-L and cleavedcaspase8/9 increased, while the protein expression of Bcl2 decreased in treatment groups. Compared with the control group, the protein expression of Bax in treatment groups had no obvious change. 3. After drug treatment, the expression of caspase3/8/9 precursor, pro-caspase3/8/9 also increased. Among them, when the concentration of saponin 6 came to 5.06 μg/m L, the expression of pro-caspase3/9 increased and differences clearly existed with statistical significance(P<0.05).Conclusion: By observing the related protein expression of extrinsic and intrinsic apoptotic pathway, saponin 6 could induce apoptosis in U87 MG cells.