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Updated: Feb 22, 2026
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Pharmacologic ascorbate induces neuroblastoma cell death by hydrogen peroxide mediated DNA damage and reduction in
Enlong Ma1, Ping Chen2, Heather M Wilkins3
1Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, USA; Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
Abstract:
An ascorbate-mediated production of oxidative stress has been shown to retard tumor growth. Subsequent glycolysis inhibition has been suggested. Here, we further define the mechanisms relevant to this observation. Ascorbate was cytotoxic to human neuroblastoma cells through the production of H2O2, which led to ATP depletion, inhibited GAPDH, and non-apoptotic and non-autophagic cell death. The mechanism of cytotoxicity is different when PARP-dependent DNA repair machinery is active or inhibited. Ascorbate-generated H2O2 damaged DNA, activated PARP, depleted NAD+, and reduced glycolysis flux. NAD+ supplementation prevented ATP depletion and cell death, while treatment with a PARP inhibitor, olaparib, preserved NAD+ and ATP levels but led to increased DNA double-strand breakage and did not prevent ascorbate-induced cell death. These data indicate that in cells with an intact PARP-associated DNA repair system, ascorbate-induced cell death is caused by NAD+ and ATP depletion, while in the absence of PARP activation ascorbate-induced cell death still occurs but is a consequence of ROS-induced DNA damage. In a mouse xenograft model, intraperitoneal ascorbate inhibited neuroblastoma tumor growth and prolonged survival. Collectively, these data suggest that ascorbate could be effective in the treatment of glycolysis-dependent tumors. Also, in cancers that use alternative energy metabolism pathways, combining a PARP inhibitor with ascorbate treatment could be useful.
Insights
High-dose vitamin C (ascorbate) induces oxidative stress, leading to neuroblastoma cell death by depleting energy and damaging DNA. Combining ascorbate with PARP inhibitors may offer new cancer treatment strategies.
Area of Science:
- Biochemistry
- Oncology
- Cell Biology
Background:
- Ascorbate-induced oxidative stress is known to inhibit tumor growth.
- The precise mechanisms, particularly concerning energy metabolism and DNA repair, require further elucidation.
Purpose of the Study:
- To define the mechanisms underlying ascorbate's cytotoxicity in human neuroblastoma cells.
- To investigate the role of poly(ADP-ribose) polymerase (PARP) in ascorbate-mediated cell death.
- To evaluate the therapeutic potential of ascorbate and PARP inhibitors in neuroblastoma.
Main Methods:
- In vitro studies using human neuroblastoma cells treated with ascorbate.
- Assessment of reactive oxygen species (ROS) production, ATP levels, NAD+ levels, and GAPDH activity.
- DNA damage assays and cell death pathway analysis (apoptosis, autophagy).
- In vivo studies using a mouse xenograft model of neuroblastoma.
Main Results:
- Ascorbate generated hydrogen peroxide (H2O2), causing ATP depletion and inhibiting GAPDH, leading to non-apoptotic cell death.
- Ascorbate-induced DNA damage activated PARP, depleting NAD+ and reducing glycolysis.
- NAD+ supplementation prevented cell death, while PARP inhibition (olaparib) preserved NAD+/ATP but increased DNA damage without preventing cell death.
- Ascorbate inhibited neuroblastoma tumor growth and prolonged survival in mice.
Conclusions:
- Ascorbate-induced cell death results from NAD+ and ATP depletion in cells with active PARP, and from ROS-induced DNA damage in the absence of PARP activation.
- Ascorbate shows potential for treating glycolysis-dependent tumors.
- Combination therapy with PARP inhibitors may be beneficial for cancers utilizing alternative energy metabolism.
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