Abstract
Radioresistance remains a significant therapeutic obstacle in glioblastoma. Reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation and apoptosis. Nox4 NADPH oxidase is abundantly expressed and has proven to be a major source of ROS production in glioblastoma. Here we investigated the effects of Nox4 on GBM tumor cell invasion, angiogenesis, and radiosensitivity. A lentiviral shRNA vector was utilized to stably knockdown Nox4 in U87MG and U251 glioblastoma cells. ROS production was measured by flow cytometry using the fluorescent probe DCFH-DA. Radiosensitivity was evaluated by clonogenic assay and survival curve was generated. Cell proliferation activity was assessed by a cell counting proliferation assay and invasion/migration potential by Matrigel invasion assay. Tube-like structure formation assay was used to evaluate angiogenesis ability in vitro and VEGF expression was assessed by MTT assay. Nox4 knockdown reduced ROS production significantly and suppressed glioblastoma cells proliferation and invasion and tumor associated angiogenesis and increased their radiosensitivity in vitro. Our results indicate that Nox4 may play a crucial role in tumor invasion, angiogenesis, and radioresistance in glioblastoma. Inhibition of Nox4 by lentivirus-mediated shRNA could be a strategy to overcome radioresistance and then improve its therapeutic efficacy for glioblastoma.
Highlights
Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor in adults with a high degree of morbidity and mortality [1]
The lentivirus-transduced cells were selected by puromycin for 10 d and the clones stably transfected with pGIPZ-lentiviral shRNAmir (Nox4-shRNA) or pGIPZ nonsilencing control vector were successfully generated
To verify that the NADPH oxidase subunit 4 (Nox4) gene was silenced by the lentivirus vector, the mRNA and protein levels in U87MG and U251 cells were assessed using real-time quantitative PCR and Western blot assays, respectively
Summary
Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor in adults with a high degree of morbidity and mortality [1]. Recent advances in the treatment of glioblastoma multiforme published in the landmark study by Stupp et al [2] changed the standard of care with the discovery that the addition of temozolomide (TMZ) to radiotherapy for newly diagnosed glioblastoma resulted in a clinically meaningful and statistically significant survival benefit with minimal additional toxicity. Despite this success, survival remains very low. Strong evidence suggests that these processes are upregulated via Nox generation of ROS
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