Combination of Ascorbic Acid and Menadione Induces Cytotoxic Autophagy in Human Glioblastoma Cells

We investigated the ability of the ascorbic acid (AA) and menadione (MD) combination, the well-known reactive oxidative species- (ROS-) generating system, to induce autophagy in human U251 glioblastoma cells. A combination of AA and MD (AA+MD), in contrast to single treatments, induced necrosis-like cell death mediated by mitochondrial membrane depolarization and extremely high oxidative stress. AA+MD, and to a lesser extent MD alone, prompted the appearance of autophagy markers such as autophagic vacuoles, autophagosome-associated LC3-II protein, degradation of p62, and increased expression of beclin-1. While both MD and AA+MD increased phosphorylation of AMP-activated protein kinase (AMPK), the well-known autophagy promotor, only the combined treatment affected its downstream targets, mechanistic target of rapamycin complex 1 (mTORC1), Unc 51-like kinase 1 (ULK1), and increased the expression of several autophagy-related genes. Antioxidant N-acetyl cysteine reduced both MD- and AA+MD-induced autophagy, as well as changes in AMPK/mTORC1/ULK1 activity and cell death triggered by the drug combination. Pharmacological and genetic autophagy silencing abolished the toxicity of AA+MD, while autophagy upregulation enhanced the toxicity of both AA+MD and MD. Therefore, by upregulating oxidative stress, inhibiting mTORC1, and activating ULK1, AA converts MD-induced AMPK-dependent autophagy from nontoxic to cytotoxic. These results suggest that AA+MD or MD treatment in combination with autophagy inducers could be further investigated as a novel approach for glioblastoma therapy.

Automated summary

In this study, the researchers investigated the ability of a combination of ascorbic acid and menadione to induce autophagy in human glioblastoma cells. The combination treatment resulted in necrosis-like cell death mediated by mitochondrial membrane depolarization and high oxidative stress. Autophagy markers were also observed, and the combination treatment affected the downstream targets of AMP-activated protein kinase, a well-known autophagy promotor. Antioxidant treatment reduced autophagy and cell death triggered by the combination treatment. Pharmacological and genetic autophagy silencing abolished the toxicity of the combination treatment, while autophagy upregulation enhanced the toxicity. These findings suggest that the combination of ascorbic acid and menadione, along with autophagy inducers, could be a potential therapeutic approach for glioblastoma.