Regulation Mechanism Of The Nrf2-ARE Signaling Pathway In Human Glioblastoma And The Effect Of The Pathway On Chemoradiotherapy Resistance

Part IERK and PI3K signaling cascades induce Nrf2activation and regulate cell viability through Nrf2in human glioblastoma cellsObjective:The extracellular regulated protein kinases (ERK) and phosphatidylinositol3-kinase (PI3K) signaling cascades are aberrantly activated in human glioblastoma cells, which results in the dysregulation of numerous downstream transcription factors. It has been reported that high levels of Nrf2promote tumor progression. Here, we investigated the effect of the ERK and PI3K signaling cascades on regulating Nrf2in human glioblastoma cells.Methods:1、The tumor tissues and the peritumoral normal tissues were collected from16patients with primary glioblastoma. We performed immunohistochemical staining to analyze the expression and distribution of Nrf2.2、In vitro, we treated the human glioblastoma cell lines with the ERK (PD9805950μM) and PI3K (LY29400210μM)inhibitors to inhibit the activation of the ERK and PI3K signaling pathway. We examined the effect of the ERK and PI3K signaling pathway on the expression and distribution of Nrf2in human glioblastoma cell lines by immunofluorescence. According to the result of immunofluorescence, we selected the U251cell line for further study. We performed western blot to investigate the effect of inhibiting the ERK and PI3K signaling pathways on the protein expression of nucleus and cytoplasm Nrf2in the U251cells. We analyzed the effect of inhibiting the ERK and PI3K signaling pathways on the mRNA expression of the Nrf2downstream genes (HO-1and NQO-1) by Real-time PCR.3、Nrf2expression was upregulated or downregulated by transient transfection. We performed MTT to analyze the effect of Nrf2on cell viability and the effect of upregulating Nrf2expression on the ERK and PI3K inhibitors-induced cell viability inhibition.Results:1、The expression and nuclear location of Nrf2are increased in human glioblastoma specimens.2、Combined inhibition of ERK and PI3K decreases the expression and transcriptional activation of Nrf2.3、The ERK and PI3K signaling cascades regulate cell viability partly through Nrf2.Conclusion:The ERK and PI3K signaling-pathways increase the expression and transcriptional activation of Nrf2and regulate cell viability partly through Nrf2in human glioblastoma. Part ⅡTemozolomide and irradiation combined treatment induces Nrf2activation in human glioblastoma cellsObjective:Resistance to chemoradiotherapy is a major obstacle to successful treatment of glioblastoma. Recently, the role of NF-E2-related factor2(Nrf2) in chemoradiotherapy resistance has been reported in several types of cancers. Here, we mainly investigated whether temozolomide and irradiation combined treatment induced Nrf2activation in human glioblastoma cells. And we further performed a preliminary study about the effect of this pathway on chemoradiotherapy resistance.Methods:1、We collected14glioblastoma patients who underwent different postoperative treatment, including untreatment (n=4), irradiation (n=5) and temozolomide plus irradiation (n=5). The effects of temozolomide and irradiation on the expression and distribution of Nrf2were evaluated by immunohistochemistry.2、In vitro, the distribution of Nrf2in human glioblastoma cell lines was examined by immunocytochemistry. Nuclear accumulation of Nrf2protein was investigated by western blot.The expression of Nrf2downstream genes were assessed by Real-time PCR.3、The effect of downregulating Nrf2expression on TMZ plus IR-induced cell death was analyzed by CCK8.Results:1、Immunohistochemical staining for Nrf2in clinical specimens showed that temozolomide and irradiation combined treatment increased the expression and nuclear accumulation of Nrf2.2、We detected increased nuclear localization of Nrf2following treatment with temozolomide plus irradiation in human glioblastoma cell lines (A172, U87,U251). Temozolomide plus irradiation increased nuclear Nrf2accumulation and induced Nrf2 target genes expression in the U251cells.3、Downregulating Nrf2expression increased TMZ plus IR-induced cell death.Conclusion:These findings suggest TMZ plus IR combined treatment induces Nrf2activation in human glioblastoma cells. The activation of Nrf2may be associate with chemoradiotherapy resistance in human glioblastoma cell. Blocking Nrf2activation may be a promising method against chemoradiotherapy resistance of glioblastoma cells.