| Part I The phenomenon of ellagic acid inhibiting the growth of glioblastoma multiforme U87 and U118 cellsObjective:To investigate the effect of ellagic acid(EA)on cell proliferation,cell apoptosis and cell invasiveness of glioblastoma multiforne(GBM)U87 and U118 cells.Methods:Cell viability was examined by cell counting kit-8(CCK-8)assay.The oncogenicity of both cell lines was measured by clone formation experiment.Cell proliferation was examined by 5-ethynyl-2'-deoxyuridine(EdU)staining.The cell cycle and cell apoptosis were measured by flow cytometry.The cell morphological changes of apoptosis were observed by Hoechst33342/PI staining.The cell transfer ability was measured by cell wound scratch assay.The cell invasion ability was examined by transwell assy.Results:The cell viability of both cell lines was inhibited by EA in a time and dose-dependent manner,after treated by EA for 48 h,the IC50 of U87 cell and U118 cells were 91.2?M and 98.6?M respectively.Clone formation assay shows the number of clone formation in experimental group is lower than control(p<0.05),which shows the oncogenicity of both cell lines were inhibited by EA effectively.We found EA could suppress the proliferation of both cell lines in EdU assay.In flow cytometry,after treated by 100?M EA for 48 h,the number of cells of S phase in U87 and U118 cells were increased by 10.89%and 11.54%respectively,accompanied by the number of cells of G1 and G2/M were decreased significantly,the difference is significant(p<0.05),which shows EA could disturb cell cycle and induce cell cycle arrest at S phase.And also we found that EA could induce cell apoptosis,after treated by 100?M EA for 48 h,the apoptosis rate in U87 and U118 cells are from(7.58± 1.13)%and(9.33±0.27)%to(23.49±0.64)%and(19.84± 1.06)%,the difference is significant(p<0.05).After stained by Hoechst33342/PI,we observed the cell morphological changes of apoptosis when exposing to EA.In cell wound scratch assay,after treated by EA for 24 h,in U87 cell lines,the wound healing rate in control and 50?M EA treated group are(51.69±0.29)%and(31.82±0.44)%;In U118 cell lines,the wound healing rate in control and 50?M EA treated group are(49.75±0.64)%and(29.82±0.69)%;the difference is significant(p<0.05),which shows EA could inhibit cell transfer ability.In transwell assay,after treated by EA for 24 h,in U87 cell lines,the cell numbers of trans-membrane in control and 100?M EA treated group are(82±3)and(31±1);In U118 cell lines,the cell numbers of trans-membrane in control and 100pM EA treated group are(97±6)and(50±4);Which shows EA could inhibit cell invasiveness.Conclusion:EA could inhibit cell proliferation,induce cell apoptosis and suppress cell invasiveness of GBM U87 and U118 cells.Part II The mechanism of ellagic acid inhibiting the growth of glioblastoma multiforme U87 and U118 cellsObjective:To investigate the specific mechanism of ellagic acid(EA)on suppressing proliferation,inducing apoptosis and inhibiting invasiveness of glioblastoma multiforme(GBM)U87 and U118 cells.Methods:DNA damage of both cell lines caused by EA exposure was evaluated by?-H2AX foci detection.The influence of EA on mitochondrial membrane potential(MMP)was detected with JC-1 Mitochondrial Membrane Potential Assay Kit.The effect of EA on activity level of MMPs in GBM cells was measured 'by gelatin zymography assay.The expression level of proliferation related genes,apoptosis related genes,invasiveness related genes in both cell lines treated with EA or without were measured by Western blot.Results:DNA damage level in experimental group was higher than control.EA could induce the changes of cell mitochondrial membrane potential in both cell lines.In gelatin zymography assay,after treated by 100?M EA for 24 h,the activity level of MMP-9 and MMP-2 of U87 cells are decreased by(78.31±2.53)%and(32.48±1.37)%;The activity level of MMP-9 and MMP-2 of U118 cells are decreased by(60.94±4.47)%and(8.99±1.02)%;the difference is significant(p<0.05),which shows EA could inhibit the activity level of MMPs in GBM cells.In Western blot analysis,we found EA could down-regulate the expression level of PCNA?Ki67?CyclinA2?Cyclin D1?CDK-2?CDK-6.EA could down-regulate the expression level of Bcl-2,and up-regulate the expression of Cleaved PARP?Bax?Active Caspase-3?DR4?DR5?And EA could up-regulate the expression of E-Cadherin,down-regulate the expression level of Akt?p-Akt?Notchl?Notch3.Conclusion:EA could inhibit cell proliferation,induce cell apoptosis and suppress cell invasiveness in both GBM cell lines by regulating Akt and Notch signaling pathway mechanically.Part ? The experimental study of ellagic acid inhibiting human glioblastoma multiforme cell growth in vivoObjective:To observe the phenomenon of ellagic acid(EA)inhibiting glioblastoma multiforme(GBM)U87 xenografts,investigate its specific mechanism and evaluate its adverse effects.Methods:Firstly,about 2×106 cells are injected subcutaneously into the flanks of Balb/c nude mice as per approved protocol;two weeks later,the tumors are taken out and cut up to establish the human GBM xenograft model for the second time.Then examine the effect of EA on tumor growth and mice weight.When tumor xenografts are dissected,HE staining is performed,and the expression of Akt and Notch signaling pathway and their target genes are detected by the immunohistochemistry(IHC).Meanwhile,visceral organs are observed,and the blood indexes are measured in order to evaluate the adverse effects of EA.Results:The inhibition rate of tumor volume in experimental group is 37.40%compared with control,the difference is significant(P<0.05),which shows EA could inhibit the growth of U87 xenografts effectively.There are large necrosis area,small inflammatory cell infiltration,hemangiectasis and congestion in experimental group after HE staining.In immunohistochemistry analysis,EA could down-regulate the expression level of PCNA?Ki67?Cyclin D1?CDK-2?CDK-6;EA could down-regulate the expression level of Bcl-2,and up-regulate the expression of Cleaved PARP?Bax?Active caspase-3?DR4?DR5;EA could up-regulate the expression of E-Cadherin,and down-regulate the expression level of Akt?p-Akt?Notchl?Notch3?Meanwhile,the body weight of the tumor bearing mice is normal,and there is no abnormity in the liver,kidney,intestine and brain of the tumor bearing mice compared with control.There is no difference in blood indexes examination between experimental and control group.Conclusion:EA could inhibit the growth of U87 xenografts by regulating Akt and Notch signaling pathways without adverse effects. |
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