High Dose Ascorbate Causes Both Genotoxic and Metabolic Stress in Glioma Cells.

Maria Castro,Melanie Mcconnell,Georgia Carson,Patries Herst

doi:10.3390/antiox6030058

Abstract

We have previously shown that exposure to high dose ascorbate causes double stranded breaks (DSBs) and a build-up in S-phase in glioblastoma (GBM) cell lines. Here we investigated whether or not this was due to genotoxic stress as well as metabolic stress generated by exposure to high dose ascorbate, radiation, ascorbate plus radiation and H2O2 in established and primary GBM cell lines. Genotoxic stress was measured as phosphorylation of the variant histone protein, H2AX, 8-oxo-7,8-dihydroguanine (8OH-dG) positive cells and cells with comet tails. Metabolic stress was measured as a decrease in NADH flux, mitochondrial membrane potential (by CMXRos), ATP levels (by ATP luminescence) and mitochondrial superoxide production (by mitoSOX). High dose ascorbate, ascorbate plus radiation, and H2O2 treatments induced both genotoxic and metabolic stress. Exposure to high dose ascorbate blocked DNA synthesis in both DNA damaged and undamaged cell of ascorbate sensitive GBM cell lines. H2O2 treatment blocked DNA synthesis in all cell lines with and without DNA damage. DNA synthesis arrest in cells with damaged DNA is likely due to both genotoxic and metabolic stress. However, arrest in DNA synthesis in cells with undamaged DNA is likely due to oxidative damage to components of the mitochondrial energy metabolism pathway.

Highlights

The last decade has seen a renewed interest in intravenous high dose ascorbate (AA) as an anticancer treatment
We analysed the extent of genotoxic and metabolic stress and the effect on DNA synthesis by high dose AA in established and patient-derived GBM cell lines and compared these effects to those of radiation, H2 O2 and combined treatments
With respect to genotoxic stress, we verified that high dose AA can generate double stranded breaks (DSBs) as well as 8OH-dG lesions

Summary

Introduction

The last decade has seen a renewed interest in intravenous high dose (pharmacological) ascorbate (AA) as an anticancer treatment. In the extracellular acidic and metal-rich tumour environment, high dose AA generates extracellular hydrogen peroxide (H2 O2 ), which diffuses into adjacent cancer cells and overwhelms the anti-oxidant defence system (reviewed by [1]). DHA is transported into cells through glucose transporters (GLUT-1), where it is reduced back to AA at the expense of glutathione (GSH), causing oxidative stress and inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycolysis, leading to ATP depletion [6,7]. In addition to causing oxidative stress, AA has been shown to increase hypoxia inducible factor, HIF-1, hydroxylase activity, leading to a decrease in HIF-1 pathway activation and a less aggressive phenotype in colorectal [8] and endometrial cancer [9], and inhibit the proliferation of breast cancer MCF-7 mammospheres [10]

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Results

Conclusion