Necrostatin-1 enhances menadione/ascorbic acid–induced oxidative stress and their cytotoxic potential in human glioblastoma U251 cell line

Abstract

The aim of this study was to investigate the role of necroptosis inhibitor necrostatin-1 (Nec-1) in death of human glioblastoma U251 cells exposed to ascorbic acid (AA), menadione(MD), and their combination, in vitro. Nec-1 augmented cytotoxicity of AA+MD, and slightly increased death of MD-treated U251 cells, as assessed by crystal violet (CV) assay. In line with previous, flow cytometric analysis of annexin/propidium iodide-stained cells showed that Nec-1 triggered cell death in MD and significantly enhanced ability of AA+MD to increase number of necrotic cells, substantiating necrosis as the mechanism of U251 cell death induced by combined treatments – AA+MD, Nec-1+MD, and Nec-1+AA+MD. Further, Nec-1 elevated mitochondrial and cellular reactive oxygen species (ROS) generated by both MD and AA+MD co-treatment, as assessed by flow cytometry analysis of MitoSOX- and DHR-stained cells, respectively. N-acetyl cysteine (NAC), a well-known antioxidant, opposed U251 cell death induced by AA+MD, Nec-1+MD, and Nec-1+AA+MD, indicating crucial role of oxidative stress in Nec-1-potentiated cytotoxicity of MD and AA+MD. Also, Nec-1 activated AMP-activated protein kinase (AMPK), and its effector molecule ULK1 (Ser317) over the level induced by MD and AA+MD, as showed by immunoblot. AMPK, highly conserved serine/threonine protein kinase, is activated under the conditions of oxidative stress probably as a consequence of depleted cellular ATP and elevated AMP levels. This result implies important role of AMPK in necrosis detected in AA+MD-, Nec-1+MD-, and Nec-1+AA+MD-treated U251 cells. Therefore, it can be concluded that ability of Nec-1 to enhance cytotoxic potential of AA+MD co-treatment and trigger cytotoxicity of MD is associated with its capacity to amplify cellular and mitochondrial ROS production, leading to necrosis-like cell death of U251 cells. Obtained results reveal potential use of Nec-1 as anti-glioblastoma agent, especially in combination with AA+MD.