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Published on: January 5, 2024
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.
Abstract:
Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (EDmax) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the EDmax was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the EDmax value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.
Insights
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.
