Methotrexate Alters Nrf2/HO‐1 Protein Expression and Intrinsic Apoptosis‐Associated Protein Responses in OVCAR‐3 Ovarian Cancer Cells: Differential Modulation by Antioxidant Compounds

ABSTRACT

Methotrexate (MTX) exerts cytotoxic effects primarily through folate pathway inhibition; however, increasing evidence suggests that MTX‐induced oxidative stress and mitochondrial apoptosis significantly contribute to cellular injury. The present study aimed to investigate MTX‐induced redox imbalance and intrinsic apoptotic activation in OVCAR‐3 ovarian cancer cells and to evaluate whether sodium selenite, fucoidan, caffeic acid, and resveratrol differentially modulate redox and mitochondrial signaling pathways. OVCAR‐3 cells were exposed for 24 h to MTX (10 µM) alone or in combination with sodium selenite (SS), fucoidan (FUC), caffeic acid (CA), or resveratrol (RSV). Oxidative stress parameters, including total antioxidant capacity (TAC), total oxidant capacity (TOC), oxidative stress index (OSI), and malondialdehyde (MDA) were measured. Redox signaling proteins (Nrf2 and HO‐1) and intrinsic apoptotic markers (Bax, Bcl‐2, cytochrome‐c, cleaved caspase‐9, and cleaved caspase‐3) were quantified by ELISA. MTX significantly decreased TAC while increasing TOC, OSI, and MDA levels, indicating marked oxidative imbalance. Concurrently, Nrf2 and HO‐1 protein expression levels were significantly reduced. MTX also increased the Bax/Bcl‐2 ratio, increased cytochrome‐c protein levels, and elevated cleaved caspase‐9 and caspase‐3 levels, consistent with activation of intrinsic mitochondrial apoptotic signaling. Antioxidant co‐treatment partially restored redox balance and attenuated alterations in intrinsic apoptosis‐associated protein markers. Among the concentrations examined, resveratrol produced the largest modulatory effect. MTX induces redox‐dependent coordinated alterations in intrinsic apoptosis‐associated proteins in OVCAR‐3 cells. Integrated evaluation of Nrf2 and HO‐1 protein expression together with cytochrome‐c–caspase signaling provides supportive evidence regarding MTX‐induced cellular stress responses. Selected antioxidants partially modulate this pathway, suggesting a regulatory role in redox‐associated chemotherapeutic stress.