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

Abstract

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.

Menadione Potentiates Auranofin-Induced Glioblastoma Cell Death

“…Finally, we decided to examine the effects of AF applied together with MEN on GBM cell viability. Cytotoxic activity of MEN, related to ROS production during its metabolism, has been documented in cancer cell lines of different origin, including GBM cells in which it was also examined in combination with ascorbic acid (AA) [ 19 , 26 , 27 ]. Nevertheless, to the best of our knowledge, there are no studies on anticancer activity of AF combined with MEN.…”

“…Moreover, we analyzed the effect of AF on the ability of patient-derived GBM cells to generate neurospheres which is considered a surrogate assay for GSC self-renewal. In addition, we studied the cellular response upon combination treatment of AF with menadione (2-methyl-1,4-naphthoquinone; MEN, vit-amin K3), whose anticancer activity is related to ROS production [ 19 ]. Furthermore, we examined the influence of treatment with AF alone or in combination with MEN on viability of normal human astrocytes (NHA).…”

Autophagy and beyond: Unraveling the complexity of UNC-51-like kinase 1 (ULK1) from biological functions to therapeutic implications

“…2 Structure 2 Activation/Inhibition Target Cell type Disease Ref. Curcumin 5-FU Inhibition AMPK/ULK1 autophagy HCT116 and HT29 colon cancer cells Colon cancer 208 Chloroquine 5-FU Activation ULK1 HCT-116 colon cancer cells Colon cancer 334 Enzalutamide 3-MA Inhibition AMPK/mTOR/ULK1 pathway J82, T24, and UMUC3 human bladder cancer cells Bladder cancer 335 Enzalutamide BAF Inhibition AMPK/mTOR/ULK1 pathway J82, T24, and UMUC3 human bladder cancer cells Bladder cancer 335 Enzalutamide CQ Inhibition AMPK/mTOR/ULK1 pathway J82, T24, and UMUC3 human bladder cancer cells Bladder cancer 335 AZD5363 FH535 Activation AMPK/mTOR/ULK1 pathway HepG2 HCC cells and Hep3B cells Hepatocellular carcinoma 336 Ascorbic acid Menadione Activation AMPK/mTORC1/ULK1 pathway Glioblastoma Glioblastoma Cells 337 …”

“…The combination of β -catenin inhibitor (FH535) and Akt inhibitor (AZD5363) enhances p53 expression and further induces lethal autophagy via regulation of the AMPK–mTOR–ULK1 pathway, exerting a more substantial effect on cell death of transformed hepatocytes, playing a treatment role in human hepatoma 336 . Combination of ascorbic acid and menadione also exerts anticancer effects through the induction of cytotoxic autophagy in human glioblastoma through the AMPK/mTOR/ULK1 pathway 337 . Curcumin pretreatment combined with 5-Fu enhanced the sensitivity of 5-Fu to colon cancer cells by inhibiting AMPK/ULK1-dependent autophagy ( Table 6 ) 208 .…”

Hirudin inhibits glioma growth through mTOR‐regulated autophagy

“…Autophagy, as a mechanism for maintaining cellular homeostasis, is an intracellular lysosomal degradation process 12 and is upregulated when stimulated by extracellular or intracellular stress to cause irreversible damage, which leads to apoptosis or necrosis 13 . Large numbers of studies have demonstrated that there is a negative correlation between autophagy activation and glioblastoma progression, 14–16 and the induction of lethal autophagy in tumour cells has been implicated in the anticancer effects of a variety of drugs. In addition, temozolomide, as a primary clinical chemotherapeutic drug for human glioma, has also been proved to play a critical role in anti‐glioblastoma therapies via autophagy induction 17,18 .…”