Antiglioma effect of ascorbic acid and menadione combination in U251 glioblastoma cell line is mediated by ROS-dependent downregulation of Akt

Abstract

Glioblastoma multiforme (GBM) represents the most common and aggressive brain tumor that still lacks effective treatment options. Tumorigenesis and progression of GBM is tightly connected with over-activation of PI3K/Akt pathway, as well as with perturbed reactive oxygen species (ROS) generation in tumor cells and microenvironment. Breaking the redox balance within the tumor cells by enhancing ROS production is one of the proposed strategies for the treatment of malignancies. The aim of this study was to investigate potential antiglioma effect of ascorbic acid (AA) and menadione (MD) combination (AA+MD), the well-known oxidative stress inducer, and determine the interplay between Akt kinase activity and ROS generation in AA+MD-treated human U251 glioblastoma cells. To this end, U251 cells were treated with AA, MD and AA+MD, in the presence or absence of antioxidant N-acetylcysteine (NAC) or selective Akt inhibitor 10-DEBC hydrochloride (DEBC). Cell viability was assessed using crystal violet and MTT assays, ROS production was evaluated by flow cytometry of dihydrorhodamine-labeled cells, while Akt activity was determined using immunoblot. In contrast to AA and MD alone, combined treatment significantly decreased viability of U251 cells. The prominent toxicity of AA+MD was accompanied by an increase in ROS generation and Akt inhibition. ROS scavenger NAC diminished both Akt inhibition and cytotoxic effect of AA+MD, suggesting that Akt inactivation and cell death induced by AA+MD are ROS-dependent. Additionally, specific Akt inhibitor DEBC further enhanced death of U251 cells and elevated AA+MD-induced ROS production. Collectively, these results suggest that PI3K/Akt serves as pro-survival pathway, and its abolishing due to excessive ROS accumulation leads to glioblastoma cell death. Further, a pro-survival role of PI3K/Akt might encompass ROS removal. In conclusion, treatment with AA and MD, particularly in combination with Akt-targeted therapy, has great potential in combating GBM which is worthy of further investigation.