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A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration
Published on: March 17, 2018
Peptide screen identifies a new NADPH oxidase inhibitor: impact on cell migration and invasion
Mohamed Mousslim1, Alessandra Pagano1, Nicolas Andreotti2
1Aix Marseille Univ, INSERM, CRO2, Faculté de Pharmacie, Marseille, France.
Abstract:
The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.
Insights
Researchers identified a new peptide, NF02, that effectively inhibits Nox1-dependent NADPH oxidase. This novel inhibitor shows potential for colorectal cancer therapy by blocking reactive oxygen species generation and cell invasion.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- NADPH oxidase (NOX) enzymes generate superoxide, a signaling molecule involved in various physiological and pathological processes.
- Nox1-dependent NOX is implicated in colorectal cancer, and its inhibition is a therapeutic strategy.
Purpose of the Study:
- To identify and characterize a novel peptide inhibitor of Nox1-dependent NADPH oxidase.
- To evaluate the specificity and efficacy of the identified peptide in inhibiting cancer cell functions.
Main Methods:
- Peptide screening using a Nox1-dependent NADPH oxidase assay in HT-29 cells.
- Electron paramagnetic resonance (EPR) to study DEPMPO adduct formation and assess superoxide scavenging activity.
- Assays to evaluate cytotoxicity, inhibition of reconstituted NOX complexes, and effects on Nox2-dependent NOX activity.
- Assessment of the peptide's impact on colorectal cancer cell migration and invasion.
Main Results:
- A cell-active peptide, NF02, was identified that potently blocks Nox1-dependent reactive oxygen species (ROS) generation.
- NF02 demonstrated no superoxide scavenging activity or impact on xanthine oxidase.
- NF02 specifically inhibited the reconstituted Nox1/Noxo1/Noxa1 complex and did not affect Nox2-dependent ROS production.
- NF02 treatment inhibited colorectal cancer cell migration and invasion.
Conclusions:
- NF02 is a novel and specific inhibitor of Nox1 over Nox2 and xanthine oxidase.
- This peptide represents a valuable tool for studying Nox1 function.
- NF02 exhibits potential therapeutic insights for colorectal cancer treatment by targeting Nox1-driven processes.
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