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Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis
Published on: January 5, 2024
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MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery.
Zhiyu Xiao1, Lingmin Xie1, Ziru Wu1
1MOE Key Laboratory of Laser Life Science & Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China.
Methods (San Diego, Calif.)
|December 6, 2025
Summary
This study developed a novel method using live-cell FRET imaging to screen cancer drugs that target the P53-MDM2 interaction. The findings identified Doxorubicin and Resveratrol as potential P53/MDM2-dependent apoptotic drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Targeting the P53-MDM2 interaction reactivates P53, a key tumor suppressor, to induce cancer cell apoptosis.
- Developing effective screening methods for drugs disrupting this interaction is crucial for cancer therapy.
Purpose of the Study:
- To establish a live-cell FRET imaging-based method for screening apoptotic drugs targeting the P53-MDM2 interaction.
- To validate the method using Nutlin-3 as a known P53-MDM2 inhibitor and to screen potential drug candidates.
Main Methods:
- Utilized live-cell Förster Resonance Energy Transfer (FRET) imaging for in situ visualization and quantitative analysis of P53-MDM2 interactions.
- Established a Nutlin-3-induced apoptosis model in MCF-7 cells, confirmed by mitochondrial membrane potential and chromatin condensation assays.
- Employed Western Blot (WB) analysis to assess BAX expression and oligomerization post-P53 activation.
Main Results:
- Nutlin-3 treatment significantly decreased P53-MDM2 interaction, evidenced by reduced FRET efficiency (EDmax from 0.50 to 0.22) and co-localization (83% to 22%).
- Nutlin-3 promoted P53 nuclear translocation and apoptosis, with BAX upregulation occurring via a Puma-independent pathway.
- Drug screening identified Doxorubicin (DOX) and Resveratrol (RSV) as potential P53/MDM2-dependent apoptotic drugs, showing decreased EDmax values of 0.29 and 0.31, respectively.
Conclusions:
- The developed live-cell FRET imaging method effectively identifies P53-MDM2 interaction inhibitors in living cells.
- DOX and RSV demonstrate potential as P53/MDM2-dependent apoptotic agents, warranting further investigation.
- This quantitative in vivo approach complements traditional drug discovery methods for cancer therapeutics targeting the P53 pathway.
