Non-genotoxic p53 reactivation via diosmin-ezetimibe-mediated MDM2 inhibition in osteosarcoma cells

• Diosmin (DIO) and Ezetimibe (EZE) synergistically reduce SJSA-1 osteosarcoma cell viability (IC₅₀ = 7.0 μM). • Combination treatment induces apoptosis with minimal necrosis, showing a safer cytotoxic profile than doxorubicin (DOX). • DIO-EZE down-regulates MDM2 and moderately elevates p53, promoting non-genotoxic p53 stabilization. • Molecular docking and 100 ns MD simulations confirm stable binding of both compounds within the MDM2 pocket. • Disruption of MDM2-p53 interaction enhances p53-mediated apoptosis, suggesting a potential non-genotoxic osteosarcoma therapy. Osteosarcoma is the most common primary bone malignancy with chemotherapy resistance leading to treatment failure and severe side effects like cardiotoxicity and myelosuppression. This research explores the combined anticancer efficacy of Diosmin (DIO), a flavonoid derived from citrus, and Ezetimibe (EZE), a cholesterol absorption blocker, against SJSA-1 osteosarcoma cells, concentrating on the modulation of the MDM2-p53 pathway. Cell viability tests indicated a dose-dependent reduction in cell survival, with an IC₅₀ of 7.0 μM for the combination, nearing the cytotoxicity of doxorubicin (DOX) at elevated doses. Morphological assessments and Annexin V/PI analyses confirmed that DIO-EZE predominantly triggered apoptosis with minimal necrosis, implying a safer cytotoxic profile in comparison to DOX. Western blot and qPCR relative gene expression analysis revealed that, in contrast to DOX, which increased p53 levels without inhibiting MDM2, the combination notably decreased MDM2 expression while moderately elevating p53, suggesting p53 stabilization via non-genotoxic pathways. Molecular docking and 100 ns molecular dynamics simulations indicated that both ligands engaged in significant hydrophobic interactions within the MDM2 binding pocket and maintained stable conformations under dynamic conditions, with EZE showing the least variation. RMSF, PCA, FEL, and per-residue energy decomposition analyses supported the structural stability and favorable binding energetics of both compounds. The results imply that DIO-EZE functions synergistically by inhibiting MDM2 expression and disrupting MDM2-p53 interactions, thereby enhancing p53 stability and facilitating apoptosis. This combination presents a promising non-genotoxic approach for p53 reactivation in osteosarcoma treatment, necessitating further mechanistic validation and in vivo studies.