The Role And Mechanism Of Nuclear Factor Nrf2-dependent Metabolisms In GBM's Cell Proliferation And Resistance To Anticancer Drugs

Glioblastoma,a highly malignant glioma,is the most aggressive primary cancer of the central nervous system and is lethal brain tumor in adults.Glioblastoma is resistant to both radiation and chemotherapy.Its invasive nature is the most challenging obstacle to surgical resection.Despite great advances in the understanding of molecular basis of glioblastomas and the advances in diagnosis,there has been limited improvement in outcomes for patients.Therefore,new target therapeutic approaches are anxiously anticipated to be explored.In response to oxidative stress,the transcription factor NF-E2-related factor 2(Nrf2)controls the fate of cells through transcriptional upregulation of antioxidant response element(ARE)-bearing genes,including those encoding endogenous antioxidants,phase ? detoxifying enzymes,and transporters.Besides mediating stress-stimulated induction of antioxidant and detoxification genes,Nrf2 can profoundly influence the cellular metabolisms.For example,Nrf2 inhibits lipogenesis,supports ?-oxidation of fatty acids,facilitates flux through the pentose phosphate pathway,and increases GSH production,NADPH regeneration and purine biosynthesis;Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis.As a result,activation of the Nrf2 pathway,by naturally-occurring compounds or synthetic chemicals at sub-toxic doses,confers protection against subsequent toxic/carcinogenic exposure.Interestingly,recent emerging data has revealed the "dark" side of Nrf2.Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues,giving cancer cells an advantage for survival and growth.Furthermore,Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance.In our study,we examined the roles of nuclear factor Nrf2 in GBM's cell proliferation and resistance to anticancer drugs.In addition,we explored how Nrf2 functioned as a tumor promoter from the new perspective of cellular metabolisms.This thesis is divided into four parts:Part ?The expression pattern of Nrf2 in GBM cell lines and its effect on cell proliferationObjective:to examine the expression pattern of Nrf2 in human glioblastoma cell lines(NHA,U251,U87,A172 and SHG44)and to investigate the potential role of nuclear factor Nrf2 in glioblastoma cell line proliferation.Methods:the expression patterns of Nrf2 in normal human astrocyte and four glioblastoma cell lines were examined by real time qPCR and western blot.The lentivirus-mediated shRNA was used to down-regulated the intracellular Nrf2.After knockdown of Nrf2,the cell proliferation was evaluated by CCK-8 method and colony formation assay.Results:All four glioma cell lines over-expressed Nrf2 than that of NHA,but the highest level was observed in U251 cells.We further evaluated the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA(shRNA).We found that Nrf2 depletion inhibited glioma cell proliferation.Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA.Conclusion:the Nrf2 was over-expressed in human glioblastoma cell lines than that of NHA,and the highest level was observed in U251 cells.The growth speed was positively related to the extent of Nrf2 activation.Silencing Nrf2 impairs glioblastoma cell proliferation.Part ?The role of Nrf2-dependent cellular energy metabolism in human GBM cell lines proliferationObjective:to investigate potential role of nuclear factor Nrf2 in glioblastoma cell line proliferation and to study the role of AMPK-activated mTOR pathway inhibition in Nrf2-depletion induced growth impairment.Methods:The intracellular ATP levels were measured using a firefly luciferase based ATP assay kit.The adenine nucleotide(AMP,ADP and ATP)pools were determined by high performance liquid chromatography(HPLC).The cell proliferation was evaluated by CCK-8 method.The status of AMPK-mTOR pathway was assessed by western blot.Results:Suppression of Nrf2 expression could lead to ATP depletion(with concomitant rise in AMP/ATP ratio)and consequently to AMPK-activated mTOR pathway inhibition.In addition,activation of adenosine monophosphate-activated protein kinase(AMPK)by treated with phenformin,an AMPK agonist,can mimic the inhibitory effect of Nrf2 knockdown in U251 cells.Conclusion:The knockdown of Nrf2 suppressed glioma cell proliferation by ATP-depletion-induced AMPK activation and consequent mTOR pathway inhibition.Part ?The role of Nrf2-dependent induction of glutathione net production in glioblastoma cell lines proliferationObjective:to investigate potential role of nuclear factor Nrf2 in glioblastoma cell line proliferation and to study the role of GSH-depletion-related AKT inhibition in Nrf2-knockdown induced growth impairment.Methods:to evaluate the biological functions of Nrf2 in U251 glioma cell(with highest Nrf2 level),the intracellular Nrf2 was specifically inhibited using short hairpin RNA(shRNA).Intracellular glutathione(GSH)level was measured by spectrophotometry.The level of intracellular reactive oxygen species was quantified using the reactive oxygen species assay kit.The cell proliferation was evaluated by CCK-8 method.The status of AKT,ERK1/2 and STAT3 pathway were evaluated by western blot.Finally,to restore Nrf2 depletion related phenotypic defects,the Nrf2 knockdown U251 cells were treated with glutathione(GSH)and N-acetylcysteine(NAC).Results:Functional analysis suggested Nrf2 depletion inhibited GBM cell proliferation and Nrf2 deficiency could also lead to oxidative stress and redox imbalance in GBM cells(diminished levels of GSH and increased levels of ROS).In addition,suppression of Nrf2 expression could also lead to AKT and ERK1/2 pathway inhibition(the phosphorylation status of STAT3 had no significant changes).Finally,we confirmed that exogenous supplementation with GSH,but not NAC,restored Nrf2 deficiency related phenotypic defects such as cell proliferation,and that GSH supplement also restored the phosphorylation status of AKT,which was impaired in Nrf2 knockdown U251 cells.Conclusions:Knockdown of Nrf2 impairs GBM cell proliferation possibly involved GSH-depletion-related AKT pathway inhibition.PartIVThe role of Nrf2-dependent induction of glutathione in temozolomide-resistant human glioblastoma cell lines U251TR and U87TRObjective:to isolate and establish in vitro two drug-resistance human glioblastoma cell line to temozolomide(named as U251TR and U87TR from U251 and U87 human glioblastoma cell line)and to investigate the potential role of nuclear factor Nrf2-dependent net GSH production in TMZ resistance of the glioblastoma cell line.Methods:Drug resistance of U87 and U251 GBM cell line was induced with escalating concentrations of TMZ to produce U251TR and U87TR.The IC50 and the drug resistance factor of U251TR and U87TR were evaluated by CCK-8 assay.Intracellular glutathione(GSH)level was measured by spectrophotometry.The expression of Nrf2 and net-GSH-production-associated enzymes were determined by western blotting.The lentivirus-mediated shRNA was used to down-regulate the intracellular Nrf2.After knockdown of Nrf2,the cytotoxicity of TMZ to U251TR and U87TR were determined by CCK-8 method.U251TR and U87TR cells were treated with exogenous GSH,the cytotoxicity and inhibition rate of TMZ to U251TR and U87TR were evaluated by CCK-8 method.Results:U251TR and U87TR showed a higher TMZ resistance than parent cell line:the RF of U251TR vs.U251 was 10.92;the RF of U87TR vs.U87 was 10.35.U251TR and U87TR also showed significantly elevated intracellular GSH level,compared parent cell line,the intracellular GSH level had increased about 3.7 and 2.4 times in U251TR and U87TR respectively.The expressions of net-GSH-production-associated enzymes,such as GCLC,GS and GR,were increased.In addition,elevated intracellular GSH level and over-expression of net-GSH-production-associated enzymes were nuclear factor Nrf2-dependent in U251TR and U87TR.Finally,we provided evidence that the depletion of Nrf2 significantly increased the cytotoxcity of TMZ to U251TR and U87TR,while exogenous supplementary GSH could impair the cytotoxcity of TMZ to U251TR and U87TR.Conclusion:the TMZ-resistant human GBM cell lines with stable resistance were successfully established by intermittent concentration gradient increment technique.The stable TMZ resistance may be related the up-regulation of Nrf2-dependent induction of glutathione.