Dual Targeting of Mitochondrial Membrane Potential and XIAP by Mebendazole Triggers Apoptosis in Ovarian Cancer Cells: An Integrated In Vitro and In Silico Perspective

Background: Ovarian cancer (OC) persists as a major cause of death in gynecological malignancies, primarily due to inherent resistance to chemotherapy and the recurrence of metastatic disease. Aim: This investigation aimed to elucidate a previously uncharacterized mechanism of action for the anthelmintic agent mebendazole (MBZ) in OC cells. Specifically, we aimed to determine if MBZ could simultaneously target mitochondrial function and X-linked inhibitor of apoptosis protein (XIAP), thereby triggering an intrinsic apoptotic pathway. Methods: In vitro studies were conducted using SKOV3 (adenocarcinoma) and PA1 (teratocarcinoma) OC cell lines. Cytotoxicity was assessed via IC 50 determination. Mitochondrial membrane potential (ΔΨm) was evaluated using flow cytometry with rhodamine-123 and confocal microscopy with JC-1 staining. Apoptosis was quantified by annexin V/PI staining. Western blot analysis was used to examine the expression of XIAP, cytochrome c, Bax, and cleaved caspase-3. Molecular docking and density functional theory (DFT) calculations were utilized to investigate MBZ’s interaction with XIAP. Results: MBZ exhibited dose-dependent cytotoxicity in both cell lines (IC 50 : 1.25 µM in SKOV3, 0.312 µM in PA1), with PA1 showing greater sensitivity. Significant ΔΨm depolarization was observed (40% in SKOV3, 33% in PA1), accompanied by increased JC-1 monomer/aggregate ratios (5.7-9.9-fold). MBZ induced apoptosis without significant necrosis (3.2-4.5-fold increase in Annexin V + cells). Mechanistically, MBZ reduced XIAP expression (2.5-fold) and upregulated cytochrome c, Bax, and cleaved caspase-3. Molecular docking revealed high-affinity binding of MBZ to the XIAP BIR3 domain, comparable with A4E (ASTX660), with unique interactions at LYS299. DFT calculations confirmed MBZ’s comparable electronic properties, including a lower Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital energy gap relative to A4E. Conclusion: This study identifies a novel dual mode of action for MBZ in OC cells, concurrently disrupting mitochondrial function and directly inhibiting XIAP, leading to caspase-dependent apoptosis. This unique mechanism of action suggests MBZ as a promising repurposed therapeutic strategy to potentially overcome chemoresistance in OC.