Research on the mechanism by which quercetin affects ferroptosis in breast cancer cells through regulating USP9X/PIAS4

Breast cancer stands as a formidable malignant tumor posing a severe threat to women’s health globally, characterized by alarmingly high rates of morbidity and mortality. Quercetin has demonstrated a substantial impact in restraining the malignant behaviors exhibited by breast cancer cells. Meanwhile, it has been uncovered that both deubiquitinating enzyme ubiquitin specific peptidase 9 X-linked (USP9X) and protein inhibitor of activated STAT 4 (PIAS4) are implicated in the progression of breast cancer. Nevertheless, thus far, no pertinent research has elucidated whether there exists an inherent association among quercetin, USP9X, and PIAS4. In this study, the viability, proliferation, and apoptosis of breast cancer cells were measured by MTT, EdU, and flow cytometry. Then, Caspase 3 activity, ROS, MDA, iron and Fe 2 + levels, and mitochondrial membrane potential were evaluated with the corresponding kits. Meanwhile, the mRNA and protein levels of USP9X and PIAS4 were determined by qRT-PCR and western blot. Besides, molecular docking was applied to visualize the binding of quercetin and USP9X. The association of USP9X with PIAS4 was predicted by bioinformatics analysis. And the ubiquitination level of PIAS4 was measured by IP and IB techniques. Firstly, quercetin treatment could effectively prevent the proliferation of breast cancer cells and promote apoptosis, oxidative stress and ferroptosis. USP9X was highly expressed in breast cancer and silencing it produced similar results as quercetin treatment. Molecular docking verified that quercetin could bind to USP9X. Meanwhile, reversion assay confirmed that quercetin inhibited the malignant behavior of breast cancer cells by binding to USP9X. At this time, it was also found that PIAS4 expression was positively correlated with USP9X, and USP9X was able to mediate the deubiquitination of PIAS4, further aggravating breast cancer. This study elucidated the mechanism of action of quercetin: by binding to USP9X, it diminished USP9X-mediated deubiquitination of PIAS4, consequently blocking the proliferation of breast cancer cells and stimulating oxidative stress and ferroptosis, ultimately achieving a therapeutic effect against breast cancer.