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Updated: Aug 25, 2025
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Manipulation of Redox Metabolism Using Pharmacologic Ascorbate Opens a Therapeutic Window for Radio-Sensitization by
Cameron M Callaghan1, Ibrahim M Abukhiran2, Amr Masaadeh2
1Department of Radiation Oncology, University of Iowa Hospital and Clinics, Iowa City, Iowa.
Purpose:
Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH-; intravenous administration achieving mM plasma concentrations) selectively enhances H2O2-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH- could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues.
Methods And Materials:
Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH-. Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy × 3) ± the ATM inhibitor KU60019 ± P-AscH-. Intestinal injury, oxidative damage, and transforming growth factor β immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH-. Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H2O2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene.
Results:
KU60019 with P-AscH- enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H2O2-dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH- markedly reduced intestinal toxicity and oxidative damage with KU60019.
Conclusions:
We provide evidence that redox modulating drugs, such as P-AscH-, may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues.
Insights
Pharmacologic ascorbate enhances ATM inhibitor radiosensitization in tumors by increasing oxidative damage. This combination protects normal tissues from radiation damage, potentially enabling clinical translation of ATM inhibitors.
Area of Science:
- Oncology
- Radiation Oncology
- Cancer Therapeutics
Background:
- Ataxia telangiectasia mutated kinase (ATM) inhibitors show promise as radiosensitizers but face clinical translation challenges due to normal tissue toxicity.
- Pharmacologic ascorbate (P-AscH⁻) selectively enhances tumor radiosensitization via H₂O₂-induced oxidative stress while protecting normal tissues as an antioxidant.
Purpose of the Study:
- To investigate if P-AscH⁻ can improve the therapeutic index of ATM inhibitor-based chemoradiation.
- To determine if P-AscH⁻ enhances ATM inhibitor efficacy in tumors and mitigates toxicity in normal tissues.
Main Methods:
- Assessed clonogenic survival in colorectal cancer cell lines and normal cells with radiation ± ATM inhibitor (KU60019) ± P-AscH⁻.
- Evaluated tumor growth delay in mouse models and quantified intestinal injury and oxidative damage after radiation ± KU60019 ± P-AscH⁻.
- Analyzed cell cycle distribution, ATM nuclear localization, and the role of H₂O₂ fluxes.
Main Results:
- KU60019 with P-AscH⁻ enhanced tumor radiosensitization through H₂O₂-dependent oxidative damage, DNA damage, G2 checkpoint abrogation, and inhibited ATM nuclear localization.
- P-AscH⁻ significantly reduced intestinal toxicity and oxidative damage when administered concurrently with KU60019.
- These effects were observed in both in vitro and in vivo colorectal cancer models.
Conclusions:
- Redox-modulating agents like P-AscH⁻ can facilitate clinical ATM inhibitor translation.
- P-AscH⁻ enhances tumor radiosensitization and protects normal tissues, improving the therapeutic index.
- This strategy offers a promising approach for combining ATM inhibitors with chemoradiation.
