The Pro-Apoptotic Effects And Mechanism Of Anemone Taipaiensis Saponin-1on Malignant Glioma U87MG And U251MG Cells

Glioma is the most commonly malignant brain tumor, which is associated with highmortality. The average survival of patients with glioma is less than18months, and the5-year survival rate less than9.8%. The current standard treatment for glioma includessurgery, radiotherapy, interstitial chemotherapy and biological therapy. Due to thecharacteristics of glioma including infiltrative growth, less possibility of completeresection, and insensitiveness to chemo-radiotherapy, recurrence of glioma is invariableand survival rate of patients is limited. Therefore, it is necessary to take further in-depthresearch and find new anticancer drugs based on inducing tumor cell apoptosis. A large number of researches indicate that Saponins extracted from natural herbs have anti-tumoractivity and potential clinical value of tumor therapy.Anemone Taipaiensis Saponins-1is a new active extract separated from AnemoneTaipaiensis, a kind of traditional Chinese medicine. Previous studies found that AnemoneTaipaiensis Saponins-1had effects of growth inhibition and apoptosis induction on avariety of tumor cells. However, it has not been reported whether it had a similar effect onglioma. Present study investigated the effects of Anemone Taipaiensis Saponins-1’s onmalignant glioma cell lines (U87MG, U251MG) and primary human brain astrocytes andits potential mechanisms, which may provide a new theoretical basis for clinicalapplication in the treatment of glioma.The content of this study is divided into four parts:Part1: The effects of Anemone Taipaiensis Saponins-1on cell growth of glioma celllines U87MG and U251MG and primary human brain astrocytes in vitro;1) Method: Applying4-methyl thiazolyl tetrazolium (MTT) assay to study the effect ofAnemone Taipaiensis Saponins-1on growth and proliferation of U87MG, U251MGand human brain astrocytes.2) Results: Anemone Taipaiensis Saponins-1significantly inhibited proliferation ofU87MG and U251MG cells in a certain time-and concentration-dependent manner;3) Conclusion: Anemone Taipaiensis Saponins-1significantly inhibited the proliferationof malignant glioma cell lines U87MG and U251MG, but had no toxic effects onastrocytes;Part2: The apoptosis induced by Anemone Taipaiensis Saponins-1in glioma cell linesU87MG, U251MG, and primary human brain astrocytes;1) Method: Applying Hoechest fluorescence staining, Annexin v-fluoresceinisothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, transmission electronmicroscopy, and the processing of the DNA Ladder assay to observe cell morphologyand apoptosis after Anemone Taipaiensis Saponins-1treatment.2) Results: Typical apoptotic morphological changes and an elevated apoptosis rate wereobserved in U87MG and U251MG cells after Anemone Taipaiensis Saponins-1 treatment.3) Conclusion: Anemone Taipaiensis Saponins-1successfully induced apoptosis inhuman brain glioblastoma cell（U87MG and U251MG）.Part3: The possible mechanism underlying Anemone Taipaiensis Saponins-1-relatedapoptosis in glioma cell lines U87MG, U251MG and primary human brain astrocytesin vitro.1) Method: Applying immunocytochemistry to observe nuclear translocation of NF-κBp65and, Western blot to observe the expression of apoptosis-related protein includingNF-κB p65, Survivin, XIAP, Bcl-2, Bax, and activation of Caspase-9, and Caspase-3after Anemone Taipaiensis Saponins-1treatment.2) Results: Anemone Taipaiensis Saponins-1induced a time-dependent induction ofapoptosis in human brain glioblastoma. At12h,24h, and72h after AnemoneTaipaiensis Saponins-1treatment, protein levels of NF-κB p65, Survivin, XIAP, andBcl-2decreased significantly, whereas the increasing of Bax and activation ofCaspase-9and Caspase-3was observed.3) Conclusion: After Anemone Taipaiensis Saponins-1treatment, the expression ofapoptosis-related proteins (e.g: NF-κB p65, Survivin, XIAP, Bcl-2, Bax) werechanged, and Caspase-9and Caspase-3were activated. Therefore we speculated thatthe effects of Anemone Taipaiensis Saponins-1on glioma cells might be associatedwith the inhibition of cell proliferation and induction of apoptosis.Part4: Anemone Taipaiensis Saponins-1for glioblastoma in nude miceexperimentally observed1） Methods: Establish U87MG and U251MG glioma cells in nude mice model byTUNEL staining in tumor cells apoptosis.2） Results: We have successfully established a nude mouse xenograft model, and foundthat Anemone Taipaiensis Saponins-1can inhibit glioblastoma U87MG and U251MGnude mice growth.3） Conclusion: In vivo successfully identified Anemone Taipaiensis Saponins-1ontransplanted tumors in nude mice significantly inhibited the proliferation of tumor cells and promote apoptosis.Anemone Taipaiensis Saponins-1can induce the apoptosis of malignant glioma celllines U87MG and U251MG; Thus, Anemone Taipaiensis Saponins-1might represent apromising therapeutic for patients with glioma in the future.