Emerging evidence indicates that modulating glutathione peroxidase 4 (GPX4) to induce ferroptosis is a promising strategy for tumor treatment. However, most of the GPX4 small molecule inhibitors face limitations due to their poor delivery efficacy and low specificity of ferroptosis activation. Herein, a ferroptosis-inducing nanomedicine is developed that integrates nutlin-3 with iridium oxide nanoclusters (NUT-IrO x NCs) for enhanced ferroptosis-driven multimodal therapeutic efficacy in colorectal cancer (CRC). This NUT-IrO x NCs can induce glutathione (GSH) depletion via enhanced Ir (VI)-Ir (III) transition, while nutlin-3, a well-established inhibitor of the p53-MDM2 interaction, suppresses GSH production by modulation of the p53/SLC7A11/xCT signaling pathway. The reduction of intracellular GSH results in pronounced reductions of GPX4 enzymatic activity, consequently leading to lipid peroxidation accumulation and further enhancing ferroptosis-induced CRC therapy. This dual-pronged approach demonstrates robust anticancer therapeutic effects with favorable biocompatibility in both in vitro and in vivo CRC models. This study provides an effective strategy that highlights the benefits of inhibiting of GSH/GPX4 by activating multiple ferroptosis regulatory pathways, providing an alternative therapeutic avenue for CRC treatment.
In breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156–214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2 ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored. Biological significance Proteins do not function as single entities but rather as a team player that has to communicate. Protein-protein interaction (PPI), normally by means of non-covalent contact among binary or large protein complex , is essential for many cellular processes including cancer progression. Thus, the ability to quantitatively and accurately monitor the changes in level of PPI with disease state can enable an improved understanding of PPI, provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI so far. The major issue of current available approaches is the trade-off between sensitivity and specificity. Thus, techniques with the ability to quantify PPIs with both high sensitivity (low false-negative rate) and high specificity (low false-positive rate) are eagerly desired. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has shown its potential to study biomolecules because of its high sensitivity, high selectivity and wide dynamic range. In this study, we made an effort to develop a LC-MS/MS-based targeted proteomics assay for the quantitative detection of p53-MDM2 interaction in breast cells and tissue samples.
Dongjuan Si,Huijuan Luo,Xiaomeng Zhang,Kundi Yang,Hongmei Wen,Wei Li,Jian Liu
Inhibition of the interactions of the tumor suppressor protein p53 with its negative regulators MDM2 in vitro and in vivo , representing a valuable therapeutic strategy for cancer treatment. The natural product chalcone exhibited moderate inhibitory activity against MDM2, thus based on the binding mode between chalcone and MDM2, a hit unsaturated pyrrolidone scaffold was obtained through virtual screening. Several unsaturated pyrrolidone derivatives were synthesized and biological evaluated. As a result, because the three critical hydrophobic pockets of MDM2 were occupied by the substituted-phenyl linked at the pyrrolidone fragment, compound 4 h demonstrated good binding affinity with the MDM2. Additionally, compound 4 h also showed excellent antitumor activity and selectivity, and no cytotoxicity against normal cells in vitro . The further antitumor mechanism studies were indicated that compound 4 h could successfully induce the activation of p53 and corresponding downstream p21 proteins, thus successfully causing HCT116 cell cycle arrest in the G1/M phase and apoptosis. Thus, the novel unsaturated pyrrolidone p53-MDM2 inhibitors could be developed as novel antitumor agents.
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