| Background: Glioma is the most common malignant brain tumor which has invasive growth;even treated by comprehensive treatment that surgery combined with postoperative radiotherapy and chemotherapy,it is difficult to be completely cured.More and more studies have shown that there were a small number of glioma stem cells exist in glioma cells,which are considered the foundation of glioma occurrence,development,maintenance,and the source of tumor recurrence and drug resistance.Therefore,drug therapy plays an important role in eliminating remaining tumor cells in surgical treatment.With the in-depth research of anti-tumor effect of traditional Chinese medicine,there are more and more people pay close attention to the antitumor effects of Chinese herbal medicine at home and abroad.Corilagin is a member of polyphenolic tannins extracted from the euphorbiaceae phyllanthus plants,it has anti-tumor effect for a variety of malignant tumor cells.NF-КB is a nuclear transcription factor which was widely found in human tissues and cellst.Our previous study showed that Corilagin can inhibit the proliferation of U251 glioma cells and U251 glioma stem cells;the inhibition on U251 glioma stem cells proliferation is stronger than U251 glioma cells.Binding to tumor stem cell theory and tumor proliferation/apoptosis theory,we research group cultivated the U251 glioma cell and stem cell lines,studied the effect of corilagin on NF-КB signaling pathway in U251 glioma cells and stem cells.In recent years,there were many literatures have been reported that corilagin have significant anti-tumor effect for a variety of malignant tumor cells,but the research of corilagin on glioma cells is relatively rare,and the research of corilagin on glioma stem cells is not yet reported.Objective: Our purpose is to explore the influence of corilagin on the the changes of morphology,the characteristics of cell proliferation,cell cycle and NF-КB signaling pathway in glioma U251 cells and stem cells,to provide new ideas and methods for the treatment of glioma.Methods: The CD133+glioma stem cells from glioma U251 cells were isolated by using immune magnetic beads.Then Nestin,GFAP and β-tubulin were used to identify glioma stem cells by Immunofluorescent assays to determine glioma stem cells’ properties.The cells were intervened by different corilagin concentrations(0,25,50,100μg/ml)for 24 h,48h and 72 h,then the changes of morphology and cell proliferation were observed by Cell Counting Kit-8(CCK-8),the cell cycle was detected and analyzed by flow cytometry,the expression of P65 gene promoter was detected by dual-luciferase reporter assay,the expression of P65 protein in nucleus and IКBα protein in cytoplasm were detected by western blot.Results:1.There is a small amount of glioma stem cells contain in glioma U251 cells,the ratio is about 0.07%.2.The glioma U251 cells grown adherently,and had intact cell structure when they were not intervened by corilagin.When they were intervened by corilagin,the cell density began to decrease with cell shrinkage and a small amount of scattered visible cell debris.With corilagin concentration and intervention time increased,the cell density decreasd greater with cell shrinkage and much amount of scattered visible cell debris.3.The glioma U251 stem cells grown suspended with spherical aggregates when they were not intervened by corilagin.When they were intervened by corilagin,the stem cell densitis began to decrease with cell shrinkage and a small amount of scattered visible cell debris.With corilagin concentration and intervention time increased,the stem cell densitis decreasd greater with stem cell shrinkage and much amount of scattered visible cell debris.4.At the same corilagin intervention time,the survival rates of glioma U251 cells and stem cells decreased with increasing of corilagin concentration;compared with the control group,the difference was statistically significant(P<0.05).At the same corilagin concentration,the survival rates of glioma U251 cells and stem cells decreased with increasing intervention time;compared with the control group,the difference was statistically significant(P<0.05).At the same corilagin intervention time and concentration,the survival rates of glioma U251 stem cells were lower than the U251 cells.5.When corilagin intervention U251 cells,the cell cycle was arrested in G2/M phase.With increasing concentrations of drug intervention,the proportion of G2/M phase increase gradually but G0/G1 phase decreased gradually;compared with the control group,the difference was statistically significant(P<0.05).When corilagin intervention U251 stem cells,the cell cycle was arrested in S phase.With increasing corilagin intervention concentrations,the proportion of S phase increase gradually but G0/G1 decreased gradually;compared with the control group,the difference was statistically significant(P<0.05).6.Adenoviral vector Ad-P65-FLUC was co-transduced with Adenoviral vector Ad-RLUC into glioma U251 cells and stem cells.At the same corilagin intervention time conditions,a lower corilagin concentration can promote P65 gene promoter expression,but with the corilagin concentration increased,the expression P65 gene promoter decreased;compared with the control group,the difference was statistically significant(P<0.05).7.When corilagin intervention glioma U251 cells and stem cells for 48 h,with the corilagin concentration increased,the expression of P65 in nuclear decreased,but IКBα in cytoplasm increased;compared with the control group,the difference was statistically significant(P<0.05).Conclusion: Corilagin could inhibit the proliferation of glioma cells and glioma stem cells;the inhibition on glioma stem-like cell proliferation was stronger than glioma cells.Corilagin could stagnate the glioma U251 cells at G2/M phase but the glioma U251 stem cells at S phase.In the NF-КB signaling pathway,corilagin might promote the expression of IКBα protein in cytoplasm and inhibit it from degradating,inhibit the expression of P65 gene promoter and P65 protein,reducing P65 protein import into the nucleus,thus inhibiting the NF-КB signaling pathway,inducing the apoptosis of tumor cells. |
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