Related Experiment Video
Updated: Mar 26, 2026
Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Published on: December 11, 2020
Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse
Jan Kučera1, Lucia Binó2, Kateřina Štefková1
1Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 267/2, 61137 Brno, Czech Republic.
Abstract:
Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.
Insights
Reactive oxygen species (ROS) regulate cellular functions. Commonly used NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium showed unexpected prooxidant effects and altered cell signaling in mouse embryonic stem cells.
Area of Science:
- Cell Biology
- Biochemistry
- Stem Cell Research
Background:
- Reactive oxygen species (ROS) are crucial regulators of cellular functions, particularly influencing embryonic stem cell differentiation.
- NADPH-dependent oxidases (NOXs) are the primary catalysts for regulated ROS production.
- Commonly used NOX inhibitors, apocynin and diphenyleneiodonium, have shown effects beyond NOX inhibition, necessitating further investigation.
Purpose of the Study:
- To investigate the potential side effects of apocynin and diphenyleneiodonium in mouse embryonic stem cells, especially given the low expression of NOXs in this model.
- To clarify the impact of these NOX inhibitors on cellular proliferation, ROS levels, and key intracellular signaling pathways.
Main Methods:
- Utilized mouse embryonic stem cells to assess the effects of apocynin and diphenyleneiodonium.
- Measured cellular proliferation and prooxidant activity using hydroethidine.
- Analyzed the impact on PI3K/Akt, Erk, and Wnt signaling pathways.
Main Results:
- Both apocynin and diphenyleneiodonium impaired cell proliferation and exhibited prooxidant activity.
- Apocynin inhibited the PI3K/Akt pathway and its downstream target Nanog, while augmenting Wnt signaling.
- Diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways, with no effect on Wnt signaling.
Conclusions:
- The study highlights limitations and unexpected interactions of NOX inhibitors with intracellular signaling pathways in embryonic stem cells.
- Apocynin and diphenyleneiodonium possess off-target effects that influence cell proliferation and signaling, independent of direct NOX inhibition.
- Findings underscore the need for careful interpretation of results obtained using these inhibitors in stem cell research.
