Establishment Of Drug-resistance Cell Line Of Human Glioma Induced By TMZ And Its Differential Gene Expression Profiles

Part one:Establishment of Drug-resistance Cell Line of Human Glioma Induced by TMZObjective Giomas can’t be cured completely by surgical resection only.Temozolomide possesses effective clinical activity against gliomas as a new methylating agent. However, the recent clinical trials show drug-resistance to temozolomide(TMZ) limits its efficacy in the therapy of gliomas and becomes a major problem in gliomas chemotherapy.Therefore ,the establishment of drug-resistance human glioma cell line induced by TMZ has significant value for the study of molecular mechanism of drug-resistance and find out the strategy to reverse gliomas drug-resistance and improve the chemotherapeutic results .Methods Chemotherapeutic resistance in U251 human glioma cells was initiated by the stepwise revulsion with TMZ. The drug-resistance index and the cell colony were assayed; We counted the cytotoxicity of TMZ to U251/TMZ by MTT method and calculated IC50 to U251/TMZ.The index of durg-resistance to U251/TMZ was calculated by regression equation method. Results was analyzed between U251/TMZ and U251.Results U251/TMZ was established by the stepwise revulsion with TMZ;The cell cloning experiment demonstrated that the colony-foming efficency of U251/TMZ-6 cells induced stepwisely with TMZ was 4~11 folds than that of U251 cells without inducement in cell culture with 20.61～164.86mM TMZ. U251/TMZ showed about 4 fold resisitance to TMZ than U251 cells assayed by MTT method.Conclusion A stable drug-resistant cell line U251/TMZ induced by TMZ was established successfully in 6-month culture,meanwhile,increasing the concentration of TMZ stepwisely was the important strategy. Part two:Research on reversion of drug-resistance in human glioma U251/TMZ by O6-BG of the inhibitor of MGMTObjective TMZ is the one of the most effective chemotheraptic drug to gliomas,however, drug-resistance limited its clinical therapeutic efficacy. As we know that MGMT play an important role in glioma drug-resistance to TMZ as a DNA repair proteinits. This study is aim to detect cytotoxic efficacy of TMZ to Mer-and Mer+ glioma cells which were pretreated by O6-BG and explore the molecular mechanism of glioma drug-resistance to TMZ.Methods The drug-resistant cell line U251/TMZ induced by TMZ and parent U251 cells were disigned into four groups:1) control group ;2) O6-BG group 3) TMZ group 4) O6-BG+TMZ group ; A standard MTT assay was used to evaluate the each group’s cytotoxicity response and the results were analyzed among the groups.Results 3.1 times of elevation of sensitivity to TMZ was observed in U251/TMZ group that was pretreated by O6-BG than it in U251 group.It shows that 10μM O6-BG treated 2 hours could obviously decrease MGMT activity of U251/TMZ cells and 50μM could completely exhausted MGMT activity in U251/TMZ cells. Meanwhile O6-BG has little toxicity to glioma cells and the survival rate was reach to 95% in 50μM concentration.Conclusion O6-BG pretreated could reverse TMZ durg-resistance in U251/TMZ cells to TMZ. It suggested that O6-BG may be a useful chemotherapeutical sensitizer combined with TMZ to gliomas.Part Three:Screen for Secondary Drug-resistant Genes in U251 cell line by cDNA MicroarrayObjective Temozolomide is widely applied as first line alkylating agent against glioma in clinical practice.And it is deemed to be one of most important strategy in glioma chemotherapy.So far only partial glioma patients could get benefit from TMZ chemotherapy.Besides the primary drug-resistant from glioma cells acquired drug-resistant also play an important role in failure of chemotherapy.To overall study the multiple molecular mechanisms of glioma drug-resistance. We employed cDNA microarray to identify differentially expressed genes between U251/TMZ and parent U251 cells.Preliminary screen for candidate drug-resistant related genes.To provide experimental clues in further understanding of molecular mechanisms of glioma drug-resistance and screening for candidate chematherapy targets.Methods Employed Agilent cDNA microarray to identify differentially expressed genes between U251/TMZ and parent cells and sceening for TMZ induced secondary drug-resistance genes by Functional Annotation Tool in DAVID Bioinformatics Resources.Results A set of secondary drug-resistant related differentially expressed gene profiles of U251/TMZ was established. Differential expression fold change≥5.0 were observed in 556 genes.22 secondary up-regulated drug-resistant related genes were identified through Functional Annotation Tool in DAVID Bioinformatics Resources.Conclusion Preliminary identified 22 up-regulated genes including ABCB4、SERPINB9、RHAG、CACNA2D1 exhibit close relationship in development of drug-resistance in U251/TMZ. It provides experimental clues in further study of molecular mechanisms of glioma drug-resistance to TMZ and the results suggested ATP-binding cassette (ABC) transporters overexpressed appears to play an important role in the development of secondary drug-resistace in U251/TMZ cells.