| Glioma,30%-40% of the intracranial tumors, can be characterized by its high proliferation rate, high recurrence rate and chemoresistance. Approximately half of all glioma in adults are glioblastoma (GB), which is the utmost lethal and frequent primary tumor in central nervous system. The prognosis of GB patients remains poor even after surgical resection and adjuvant radiotherapy and chemotherapy, mainly because of the rapid and infiltrative growth, the high recurrence rate, and the strong resistance to treatment. Thus, it is necessary to take great efforts in looking for new molecular markers and targeted therapies, in order to achieve earlier diagnosis and improve the prognosis. Nuclear factor E2-related factor 2 (Nrf2) has the potential to be one of the targeted genes in gene-target therapy.Nrf2 plays a central role in regulating intracellular antioxidants and phase Ⅱ detoxification enzymes. As a transcription factor, Nrf2 belongs to the Keap 1-Nrf2-ARE (antioxidant response element) signaling pathway, and upregulates many ARE-containing genes. Since being found, Nrf2 has been confirmed to help protect normal cells from harmful stimulus, including inflammation, trauma, ischemia, hemorrhage and cancer. However, recently there is increasing concern regarding the deleterious role of the Nrf2 in cancer cell biology. That is, high level of Nrf2 promoted cancer cell proliferation, tumor growth and contributed to chemoresistance of cancer cells. Our preliminary study has also demonstrated that Nrf2 was involved in cell apoptosis, autophagy, migration and invasion of glioma cell U251. Based on these results, we hypothesized that Nrf2 plays a pivotal role in glioblastoma cell proliferation, tumor growth and angiogenesis.We examined the expression of Nrf2, CD31and HIF-1 a in GB specimens by tissue microarray and immunohistochemistry, and correlated this investigation to the outcome of GB patients. The protein level of Nrf2 was positively correlated with the MVD value assessed by CD31. Nrf2 and HIF-1α were overexpressed in GB tissues. There was significant correlation between HIF-1α level and Nrf2 status. Kaplan-Meier analysis showed high Nrf2 expression was significantly associated with shorter overall survival (OS), and was identified as an independent prognostic factor in multivariate analysis.Then we contructed the plasmid Si-Nrf2 and tranduced it into the U251 cells to downregulate the expression of Nrf2 and established the stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed the cell proliferation in vitro and tumor growth in mouse xenograft models. After that we performed the immunohistochemistry staining to detect the protein levels of Nrf2, Ki67, Caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki67 were much lower in group Si-Nrf2 than group Si-control. And the number of Caspase-3 positive cells was significantly increased in group Si-Nrf2. In the analysis of microvessel density (MVD) assessed by CD31, MVD value in group Si-Nrf2 decreased significantly compared to group Si-control.In the next part we continued to culture the stable Nrf2-knockdown cells and mimicked hypoxic condition in vitro. The mRNA and protein levels were quantified by real-time PCR and Western blotting. The supernatants of cell cultures were collected to assess the VEGF expression by ELISA and to perform endothelial tube formation assays. Tumor vessel formation were assessed in mouse xenografts. In Nrf2-knockdown cell lines in hypoxia, the mRNA and protein levels of downgstream genes were lower than the control, and notably less HIF-1α protein was accumulated, with a lower expression of VEGF. The downregulation of Nrf2 also suppressed the vessel formation in endothelial tube formation assays and mouse xenografts. Yet the mRNA level of HIF-1α was not affected by the different expression levels of Nrf2.To conclude, Nrf2 and HIF-1α are associated with each other in GB and high Nrf2 expression is associated with poor outcome of GB patients. Nrf2, as a critical transcription factor, plays a candidate role in controlling glioma angiogenesis, probably by affecting the protein level of HIF-1α expression. All these results highlight Nrf2 as a candidate molecular target to control the GB cell proliferation, tumor growth, and angiogenesis. |
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