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.

Methotrexate (MTX) altered oxidative stress parameters, reduced Nrf2 and HO‐1 protein expression, increased intrinsic apoptosis‐associated protein markers, and decreased cell viability in OVCAR‐3 cells. Antioxidant co‐treatment partially modulated these protein responses and oxidative stress parameters. Among the concentrations examined, resveratrol produced the largest modulatory effects.

1. Introduction

Ovarian cancer remains one of the most lethal gynecological malignancies worldwide, largely due to late‐stage diagnosis, aggressive tumor biology, and the emergence of chemoresistance [1, 2]. Although cytoreductive surgery combined with chemotherapy constitutes the standard treatment approach, therapeutic efficacy is frequently compromised by tumor‐associated stress responses, redox imbalance, and dysregulation of apoptosis signaling pathways [3, 4]. Increasing evidence suggests that beyond direct cytotoxicity, the cellular redox state critically influences chemotherapy responsiveness and cell fate determination in ovarian cancer.

Methotrexate (MTX) is a folate antagonist that inhibits dihydrofolate reductase, thereby impairing nucleotide biosynthesis and suppressing DNA replication [5, 6]. While its primary mechanism involves disruption of folate‐dependent metabolic pathways, accumulating data indicate that MTX‐induced cytotoxicity extends beyond cell cycle arrest and includes excessive generation of reactive oxygen species (ROS). Disturbance of NADPH‐dependent redox homeostasis following folate pathway inhibition has been proposed as a key contributor to oxidative stress during MTX exposure [7]. Elevated ROS levels promote lipid peroxidation, protein oxidation, and oxidative DNA damage, ultimately triggering mitochondrial dysfunction and apoptosis [8, 9].

The intrinsic (mitochondrial) apoptotic pathway is tightly regulated by members of the Bcl‐2 protein family. A shift in the balance between pro‐apoptotic proteins such as Bax and anti‐apoptotic proteins such as Bcl‐2 increases mitochondrial outer membrane permeability, facilitating the release of cytochrome‐c into the cytosol [10]. Released cytochrome‐c then participates in apoptosome formation, leading to activation of caspase‐9 and subsequent cleavage of executioner caspase‐3. In this context, the Bax/Bcl‐2 ratio is widely used as a functional indicator of apoptotic susceptibility [11, 12]. However, evaluation of Bax/Bcl‐2 alterations alone may not fully capture the dynamic activation of the intrinsic apoptotic cascade, which requires confirmation at the level of mitochondrial signaling intermediates and initiator caspases.

In parallel with apoptotic regulation, the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway plays a central role in maintaining cellular redox homeostasis. Under oxidative stress conditions, Nrf2 dissociates from its cytoplasmic repressor Keap1 and translocates to the nucleus, where it induces the expression of antioxidant response element (ARE)‐driven genes, including heme oxygenase‐1 (HO‐1) [13]. Dysregulation of the Nrf2/HO‐1 axis has been implicated in chemotherapy‐induced oxidative damage as well as in cancer cell survival and drug resistance mechanisms [14]. In ovarian cancer, altered redox adaptation mechanisms may critically influence the balance between cytotoxicity and survival during chemotherapeutic exposure.

The modulation of chemotherapy‐induced oxidative and apoptotic responses by antioxidant compounds has attracted considerable attention in recent years. Sodium selenite contributes to endogenous antioxidant defense through its role in selenoprotein synthesis [15, 16]. Fucoidan, a sulfated polysaccharide, has been reported to exert antioxidant and apoptosis‐modulating effects [17, 18]. Polyphenolic compounds such as caffeic acid and resveratrol demonstrate ROS‐scavenging capacity and can influence mitochondrial integrity and apoptosis‐related signaling pathways [19, 20, 21]. However, whether these agents modulate MTX‐induced redox imbalance through regulation of upstream antioxidant signaling pathways and downstream mitochondrial apoptotic mediators in ovarian cancer cells remains insufficiently characterized.

Most previous in vitro studies have either focused on single antioxidant compounds or predominantly evaluated transcriptional alterations without comprehensive protein‐level confirmation of redox and mitochondrial signaling events [22, 23]. Given that apoptosis execution and redox adaptation are primarily regulated at the protein level, integrated evaluation of oxidative stress markers together with key mediators of redox regulation and intrinsic apoptotic signaling may provide a more informative framework for understanding MTX‐induced cellular stress responses.

The antioxidant compounds included in the present study were selected to represent distinct classes of antioxidant agents with different biological characteristics. By comparing compounds with different molecular characteristics within the same experimental model, we sought to determine whether modulation of MTX‐induced oxidative stress and apoptotic signaling differs according to antioxidant class and mechanism of action.

Therefore, the aim of the present study was to investigate MTX‐induced oxidative stress and intrinsic mitochondrial apoptotic signaling in OVCAR‐3 ovarian cancer cells and to evaluate whether sodium selenite, fucoidan, caffeic acid, and resveratrol differentially modulate redox‐associated and apoptosis‐related protein responses under MTX exposure.

2. Materials and Methods

2.1. Chemicals

Methotrexate (MedChem, USA; Cat. No.: HY‐14519) was used to establish the experimental cellular stress model. Antioxidant agents included sodium selenite (Sigma‐Aldrich, USA; Cat. No.: 214485‐5G), fucoidan (MedChem, USA; Cat. No.: HY‐132179), caffeic acid (MedChem, USA; Cat. No.: HY‐N0172), and resveratrol (MedChem, USA; Cat. No.: HY‐16561). Methotrexate, caffeic acid, and resveratrol were dissolved in DMSO, whereas sodium selenite and fucoidan were dissolved in sterile distilled water (dH₂O). Stock solutions were prepared according to the manufacturers' recommendations and diluted in culture medium immediately before use. Vehicle control conditions corresponding to each solvent were included in the experimental design. For compounds dissolved in DMSO, the final DMSO concentration in the culture medium did not exceed 0.1% (v/v).

2.2. Cell Line and Culture Conditions

OVCAR‐3 human ovarian adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; HTB‐161). Cells were cultured according to ATCC recommendations and routinely monitored for mycoplasma contamination. No additional STR‐based cell line authentication was performed following receipt from the certified cell repository. OVCAR‐3 cells were maintained in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin‐streptomycin, and 1% l‐glutamine under standard culture conditions (37°C, 5% CO₂). The medium was routinely changed every 2–3 days to support optimal cell growth.

All experiments were performed using cells in the logarithmic growth phase. At the beginning of each experiment, cell confluency was adjusted to approximately 70%–80%. To minimize biological variability, experiments were conducted using cells at similar passage numbers, and all treatments were carried out under identical culture conditions.

2.3. Experimental Groups, Drug Concentrations, and Treatment Protocol

Cells were randomly assigned to the following experimental groups:

Group 1. Control: Cells treated with vehicle only

Group 2. MTX: Cells treated with methotrexate (MTX, 10 µM)

Group 3. MTX + Sodium selenite (SS): Cells were exposed to MTX (10 µM) together with sodium selenite (100 nM)

Group 4. MTX + Fucoidan (FUC): Cells were exposed to MTX (10 µM) in combination with fucoidan (50 µg/mL)

Group 5. MTX + Caffeic acid (CA): Cells were exposed to MTX (10 µM) in combination with caffeic acid (50 µM)

Group 6. MTX + Resveratrol (RSV): Cells were exposed to MTX (10 µM) in combination with resveratrol (25 µM)

Based on preliminary MTT viability assessment and published literature, MTX was administered at a concentration that produced substantial but incomplete cytotoxicity, thereby allowing evaluation of oxidative stress‐ and apoptosis‐associated signaling responses [24, 25]. Sodium selenite, fucoidan, caffeic acid, and resveratrol were applied at concentrations commonly used in in vitro studies and reported to exert antioxidant and cytoprotective effects [26, 27, 28, 29]. All antioxidant compounds were co‐administered simultaneously with MTX, and cells were incubated for 24 h under standard culture conditions. All experimental procedures were performed in parallel to ensure consistency across groups.

2.4. Metabolic Viability (MTT Assay)

MTT metabolic activity was assessed using the MTT assay. Briefly, OVCAR‐3 cells were seeded into 96‐well plates and allowed to attach overnight under standard culture conditions. Following treatment with MTX alone or in combination with sodium selenite, fucoidan, caffeic acid, or resveratrol for 24 h, MTT reagent (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) was added to each well and incubated for 4 h at 37°C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader. MTT assay was expressed as a percentage of the untreated control group, which was defined as 100%.

2.5. Cell Lysate Preparation

After 24 h of treatment, cells were washed twice with ice‐cold phosphate‐buffered saline (PBS) and lysed using an appropriate lysis buffer without detergents. Cell lysates were homogenized and clarified by centrifugation at 12,000 g for 10 min at 4°C to eliminate cellular debris. The resulting supernatants were subsequently collected for oxidative stress measurements and protein analyses. Total protein concentrations of cell lysates were determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Rockford, IL, USA). All samples were normalized to equal protein concentrations prior to ELISA measurements and biochemical analyses [30].

2.6. Measurement of Oxidative Stress Markers

Malondialdehyde (MDA) levels in cell lysates were quantified using a commercially available colorimetric MDA Assay Kit (Abbexa Ltd, Cambridge, UK; Catalog No. abx294023), which is based on the thiobarbituric acid (TBA) reaction. In this assay, MDA in the sample reacts with TBA to form a colored adduct measurable at 532 nm using a microplate reader. Cell lysates prepared as described above were mixed with the extraction and chromogenic reagents according to the manufacturer's protocol, incubated, and the absorbance was determined at 532 nm. MDA levels were quantified based on a standard calibration curve and normalized to total protein content (nmol/mg protein).

Total antioxidant capacity (TAC) was determined in cell lysates using a commercially available colorimetric assay kit (Rel Assay Diagnostics, Türkiye) based on the Trolox equivalent antioxidant capacity principle, following the manufacturer's protocol. TAC results were reported as mmol Trolox equivalent per mg protein.

Total oxidant capacity (TOC) was determined using a colorimetric assay kit (Rel Assay Diagnostics, Türkiye), which measures the oxidation of ferrous ion to ferric ion by oxidants present in the sample. Results were calibrated with hydrogen peroxide standards and expressed as µmol H₂O₂ equivalent per mg protein.

The oxidative stress index (OSI) was calculated as [(TOC/TAC) × 100] arbitrary units.

2.7. Measurement of Redox Signaling Proteins

To evaluate the redox response induced by MTX administration, Nuclear factor erythroid 2‐related factor 2 (Nrf2, Human NFE2L2 (Nrf2) ELISA Kit, ELK Biotechnology, Cat No: ELK6819, China) and heme oxygenase‐1 (Human HO‐1 ELISA Kit, ELK Biotechnology, Cat No: ELK1476, China) protein levels in cell lysates were measured using commercially available enzyme‐linked immunosorbent assay (ELISA) kits. Measurements were performed according to the manufacturer's instructions. Briefly, prepared cell lysate samples were added to microtiter plates pre‐coated with specific capture antibodies and labeled with an appropriate enzyme‐conjugated secondary antibody after incubation. The resulting color reaction after the addition of the substrate solution was measured at the appropriate wavelength using a microplate reader. Absorbance values for each protein were converted to quantitative values using standard curves. Nrf2 and HO‐1 concentrations were expressed as ng/mL based on the standard calibration curves provided by the manufacturer. All analyses were performed with at least three independent biological replicates.

2.8. Measurement of Apoptotic Markers

Cellular apoptotic responses were evaluated in cell lysates by quantifying Bax (Human BAX ELISA Kit, Abcam, Cat No: ab199080, UK), Bcl‐2 (Human Bcl‐2 ELISA Kit, Abcam, Cat No: ab119506, UK), cleaved caspase‐3 (Human Cleaved Caspase‐3 (Asp175) ELISA Kit, Abcam, Cat No: ab220655, UK), cleaved caspase‐9 Human Cleaved Caspase‐9 ELISA Kit, Antibodies.com, Cat No: A73890, UK), and cytochrome‐c (Human Cytochrome‐c ELISA Kit, Abcam, Cat No: ab221832, UK) protein levels using commercially available enzyme‐linked immunosorbent assay (ELISA) kits, in accordance with the manufacturers' instructions.

Briefly, aliquots of prepared cell lysates were added to microplate wells precoated with specific capture antibodies and incubated under appropriate conditions. Bound proteins were subsequently detected using enzyme‐conjugated secondary antibodies, followed by substrate development. The resulting colorimetric reaction was measured at the recommended wavelength for each analyte using a microplate reader. Absorbance values were converted into quantitative protein concentrations based on standard calibration curves.

Protein concentrations were expressed as pg/mL according to the standard curves. To assess apoptotic balance, the Bax/Bcl‐2 ratio was calculated individually for each sample and used as a principal output parameter reflecting susceptibility to apoptosis. Cytochrome‐c protein levels were interpreted as an intrinsic apoptosis‐associated protein marker, while cleaved caspase‐9 and cleaved caspase‐3 were considered markers of initiator and executioner caspase activation, respectively. Together, these proteins were evaluated to characterize intrinsic apoptosis‐associated protein responses. Enzyme‐linked immunosorbent assays have been widely employed for the quantitative determination of apoptosis‐associated proteins in cell lysates, including active caspase forms, as reliable indicators of programmed cell death [11].

2.9. Statistical Analysis

All experiments were performed using three independent biological replicates. Each biological replicate represents an independently conducted cell culture experiment performed on a different occasion. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Data normality was examined using the Shapiro–Wilk test, which confirmed a normal distribution. Accordingly, results are reported as mean ± standard deviation (SD). Differences among multiple experimental groups were analyzed by one‐way analysis of variance (ANOVA). In cases where a significant overall effect was observed, Tukey's post hoc test was employed for pairwise comparisons. Statistical significance was defined as p < 0.05.

3. Results

3.1. Effects of Methotrexate and Antioxidant Co‐Treatments on MTT Metabolic Activity

Metabolic viability was evaluated using the MTT assay after 24 h of treatment (Figure 1). Compared with the control group, MTX significantly reduced metabolic viability (53 ± 6% vs. 100 ± 4%, p < 0.001), indicating substantial cytotoxicity under the experimental conditions. Co‐treatment with antioxidant compounds significantly improved MTT metabolic activity compared with MTX alone (p < 0.01 for all comparisons). Among the concentrations examined, the largest increase in MTT metabolic activity was observed in the MTX + RSV group (89% ± 3%), followed by MTX + CA (83% ± 4%), MTX + FUC (74% ± 2%), and MTX + SS (68% ± 5%). Nevertheless, viability remained lower than control values in all treatment groups. These findings indicate that antioxidant co‐treatment partially attenuated MTX‐associated loss of MTT metabolic activity.

Figure 1.

Effects of methotrexate and antioxidant co‐treatment on metabolic viability (MTT assay) in OVCAR‐3 cells. MTT metabolic activity was assessed using the MTT assay after 24 h of treatment with methotrexate (MTX) alone or in combination with sodium selenite (SS), fucoidan (FUC), caffeic acid (CA), or resveratrol (RSV). Metabolic viability (MTT assay) values are expressed as percentages relative to the untreated control group. Data are presented as mean ± SD from three independent biological replicates. Each biological replicate represents an independently conducted cell culture experiment performed on a separate occasion. MTX significantly reduced MTT metabolic activity compared with the control group, whereas co‐treatment with antioxidant compounds partially restored viability. Among the concentrations examined, resveratrol produced the largest increase in MTT metabolic activity, followed by CA, FUC, and SS. Different lowercase letters above the bars indicate statistically significant differences among groups (one‐way ANOVA followed by Tukey's post hoc test, p < 0.05).

3.2. Effects of Methotrexate on Oxidative Stress Parameters

Oxidative stress status in OVCAR‐3 cells was evaluated by measuring TAC, TOC, OSI, and malondialdehyde (MDA) levels (Figure 2).

Figure 2.

Effects of methotrexate and antioxidant co‐treatments on oxidative stress parameters in OVCAR‐3 cells. (A) Total antioxidant capacity (TAC); (B) total oxidant capacity (TOC); (C) oxidative stress index (OSI); and (D) malondialdehyde (MDA) levels. TAC, TOC, OSI, and MDA levels were measured in OVCAR‐3 cells after 24 h of treatment with methotrexate (MTX) alone or in combination with sodium selenite (SS), fucoidan (FUC), caffeic acid (CA), or resveratrol (RSV). Oxidative stress parameters were quantified using commercially available assay kits according to the manufacturers' instructions. Data are presented as mean ± SD from three independent biological replicates. Each biological replicate represents an independently conducted cell culture experiment performed on a separate occasion. Different lowercase letters indicate statistically significant differences among groups (one‐way ANOVA followed by Tukey's post hoc test, p < 0.05).

Compared with the control group, MTX treatment resulted in a significant reduction in TAC levels (0.72 ± 0.08 vs. 1.25 ± 0.10 mmol Trolox eq./mg protein, p < 0.001). In parallel, TOC levels were markedly elevated in the MTX group compared with controls (12.40 ± 1.10 vs. 6.20 ± 0.55 µmol H₂O₂ eq./mg protein, p < 0.001). Consistent with this oxidant shift, intracellular MDA levels significantly increased following MTX exposure (4.85 ± 0.40 vs. 2.10 ± 0.18 nmol/mg protein, p < 0.001). As a result, the OSI value was substantially elevated in MTX‐treated cells relative to controls (17.22 ± 1.95 vs. 4.96 ± 0.48 arbitrary units, p < 0.001).

Co‐treatment with antioxidant agents significantly improved oxidative stress parameters compared with MTX alone (p < 0.01 for all comparisons). TAC levels increased in all antioxidant groups, with the highest value among the concentrations examined in the MTX + RSV group (1.12 ± 0.09 mmol Trolox eq./mg protein). Concurrently, TOC levels were significantly reduced, reaching 7.40 ± 0.70 µmol H₂O₂ eq./mg protein in the MTX + RSV group. Similarly, MDA levels decreased across antioxidant‐treated groups, with the lowest level among the concentrations examined observed in the RSV group (2.65 ± 0.22 nmol/mg protein).

Consistent with these changes, OSI values were significantly attenuated in all antioxidant co‐treatment groups compared with MTX alone (p < 0.01). Among the concentrations examined, the largest reduction was observed in the MTX + RSV group (6.61 ± 0.72), although OSI values remained higher than those of the untreated control group (p < 0.05).

3.3. Modulation of Nrf2/HO‐1 Redox Signaling

To determine whether MTX‐induced oxidative stress was associated with alterations in redox regulatory pathways, Nrf2 and HO‐1 protein levels were quantified in OVCAR‐3 cell lysates using ELISA (Figure 3).

Figure 3.

Effects of methotrexate and antioxidant co‐treatments on Nrf2 and HO‐1 protein levels in OVCAR‐3 cells. (A) Nrf2 protein levels and (B) HO‐1 protein levels. Nrf2 and HO‐1 protein levels were measured in OVCAR‐3 cells after 24 h of treatment with methotrexate (MTX) alone or in combination with sodium selenite (SS), fucoidan (FUC), caffeic acid (CA), or resveratrol (RSV). Protein concentrations were quantified by ELISA following normalization of total protein content using the bicinchoninic acid (BCA) assay. Data are presented as mean ± SD from three independent biological replicates. Each biological replicate represents an independently conducted cell culture experiment performed on a separate occasion. Different lowercase letters indicate statistically significant differences among groups (one‐way ANOVA followed by Tukey's post hoc test, p < 0.05).

Compared with the control group, MTX treatment resulted in a significant reduction in Nrf2 protein levels (0.98 ± 0.12 vs. 2.35 ± 0.21 ng/mL, p < 0.001). A similar decrease was observed in HO‐1 expression, with MTX‐treated cells exhibiting markedly lower HO‐1 levels than controls (1.25 ± 0.14 vs. 3.10 ± 0.28 ng/mL, p < 0.001). These findings indicate that MTX exposure is associated with reduced Nrf2 and HO‐1 protein expression levels in OVCAR‐3 cells.

Co‐treatment with antioxidant agents significantly increased Nrf2 and HO‐1 protein levels compared with MTX alone (p < 0.01 for all comparisons). Among the concentrations examined, resveratrol produced the largest increase in Nrf2 and HO‐1 protein expression, elevating Nrf2 levels to 2.05 ± 0.18 ng/mL and HO‐1 levels to 2.75 ± 0.25 ng/mL (p < 0.05 vs. sodium selenite and fucoidan groups).

Although antioxidant co‐treatment partially restored Nrf2 and HO‐1 expression toward control values, protein levels in all co‐treatment groups remained modestly lower than those observed in untreated control cells (p < 0.05), suggesting incomplete recovery of redox signaling under the present experimental conditions.

Collectively, these findings indicate that MTX‐induced oxidative imbalance in OVCAR‐3 cells is associated with reduced Nrf2 and HO‐1 protein expression, and that antioxidant compounds differentially modulate this redox regulatory system at the protein level.

3.4. MTX‐Induced Activation of the Intrinsic Mitochondrial Apoptotic Pathway

To further elucidate whether MTX‐induced oxidative imbalance translated into activation of the intrinsic mitochondrial apoptotic pathway, Bax, Bcl‐2, cytochrome‐c, cleaved caspase‐9, and cleaved caspase‐3 protein levels were quantified in OVCAR‐3 cells (Figure 4).

Figure 4.

Effects of methotrexate and antioxidant co‐treatments on intrinsic apoptosis‐associated protein levels in OVCAR‐3 cells. Protein levels of (A) Bax, (B) Bcl‐2, (C) cytochrome‐c, (D) cleaved caspase‐9, (E) cleaved caspase‐3, and (F) the Bax/Bcl‐2 ratio were measured by ELISA in OVCAR‐3 cells after 24 h of treatment with methotrexate (MTX) alone or in combination with sodium selenite (SS), fucoidan (FUC), caffeic acid (CA), or resveratrol (RSV). Protein concentrations were quantified following normalization of total protein content using the bicinchoninic acid (BCA) assay. The Bax/Bcl‐2 ratio was calculated individually for each sample. Data are presented as mean ± SD from three independent biological replicates. Each biological replicate represents an independently conducted cell culture experiment performed on a separate occasion. Different lowercase letters indicate statistically significant differences among groups (one‐way ANOVA followed by Tukey's post hoc test, p < 0.05).

Compared with the control group, MTX treatment significantly increased Bax protein levels (260.38 ± 22.11 vs. 120.45 ± 10.32 pg/mL, p < 0.001), while Bcl‐2 expression was markedly reduced (115.42 ± 10.18 vs. 240.18 ± 18.44 pg/mL, p < 0.001). Consequently, the Bax/Bcl‐2 ratio was significantly elevated in MTX‐treated cells (2.28 ± 0.25 vs. 0.50 ± 0.06, p < 0.001), indicating a pronounced shift toward a pro‐apoptotic profile.

Cytochrome‐c protein levels were significantly higher in the MTX group compared with controls (185.3 ± 16.8 vs. 82.6 ± 7.4 pg/mL, p < 0.001). Increased cytochrome‐c protein levels were accompanied by significantly higher cleaved caspase‐9 levels (125.6 ± 11.9 vs. 38.4 ± 4.6 pg/mL, p < 0.001) and cleaved caspase‐3 levels (165.63 ± 14.27 vs. 55.27 ± 6.15 pg/mL, p < 0.001), together with an increased Bax/Bcl‐2 ratio. Collectively, these findings indicate coordinated alterations in intrinsic apoptosis‐associated protein markers following MTX exposure.

In all antioxidant co‐treatment groups, cytochrome‐c levels were significantly reduced compared with MTX alone (p < 0.01 for all comparisons). Among the concentrations examined, the largest attenuation was observed in the MTX + RSV group (95.4 ± 8.6 pg/mL), although values remained modestly higher than those of the control group (p < 0.05). Similarly, cleaved caspase‐9 and cleaved caspase‐3 levels were significantly decreased in antioxidant‐treated groups relative to the MTX group (p < 0.01).

Parallel to these changes, Bax expression was significantly lower and Bcl‐2 expression significantly higher in all antioxidant co‐treatment groups compared with MTX‐treated cells (p < 0.01). As a result, the Bax/Bcl‐2 ratio was significantly reduced, with the lowest ratio observed in the MTX + RSV group (0.87 ± 0.10, p < 0.05 vs. sodium selenite and fucoidan groups).

Collectively, these data indicate that MTX induces robust activation of the mitochondrial apoptotic pathway in OVCAR‐3 cells, characterized by Bax/Bcl‐2 imbalance, increased cytochrome‐c protein levels, initiator caspase‐9 activation, and downstream caspase‐3 cleavage. Antioxidant co‐treatment partially attenuated alterations in intrinsic apoptosis‐associated protein markers. Among the concentrations examined, resveratrol produced the largest modulatory effect.

4. Discussion

In the present study, MTX‐induced redox imbalance and intrinsic mitochondrial apoptotic responses were comprehensively evaluated at the protein level in the human ovarian serous carcinoma cell line OVCAR‐3. Furthermore, the modulatory effects of selected antioxidant agents were examined within the context of redox‐ and apoptosis‐associated signaling responses. The findings demonstrate that MTX exposure not only exacerbates oxidative stress parameters but also is associated with reduced Nrf2 and HO‐1 protein expression and is associated with coordinated alterations in intrinsic apoptosis‐associated protein markers.

Although the cytotoxic effects of MTX have traditionally been attributed to inhibition of folate metabolism and suppression of DNA synthesis [5, 6], accumulating evidence indicates that MTX also disrupts cellular redox homeostasis, leading to excessive generation of ROS [31, 32]. Inhibition of the folate pathway may reduce NADPH production and compromise antioxidant defense systems, thereby rendering cells more susceptible to oxidative damage [33]. In the current study, the marked decrease in TAC and the significant increases in TOC and MDA levels in the MTX‐treated group support the presence of a pronounced oxidative shift in OVCAR‐3 cells. These findings are consistent with previous reports linking ROS overproduction to lipid peroxidation and cellular injury [8, 9].

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a central regulator of cellular redox homeostasis. Under physiological conditions, Nrf2 is sequestered in the cytoplasm by its repressor Keap1; however, under oxidative stress, it translocates to the nucleus and activates antioxidant response element (ARE)‐driven genes, including heme oxygenase‐1 (HO‐1) [16, 34]. Nevertheless, excessive or sustained oxidative stress may impair or overwhelm the Nrf2 response [35]. In this study, MTX significantly reduced Nrf2 and HO‐1 protein levels, suggesting compromised redox adaptive capacity in OVCAR‐3 cells. Partial restoration of Nrf2 and HO‐1 expression following antioxidant co‐treatment indicates that redox signaling can be modulated under MTX‐induced stress conditions. Among the concentrations examined, resveratrol produced the largest increase in Nrf2 and HO‐1 protein expression on the Nrf2/HO‐1 axis, consistent with previous studies demonstrating its pleiotropic influence on redox‐sensitive transcriptional pathways [20, 21].

Redox imbalance is well recognized as a trigger of mitochondrial apoptotic signaling. Members of the Bcl‐2 protein family are key regulators of mitochondrial outer membrane integrity [36, 37]. In the present study, MTX exposure led to increased Bax levels and decreased Bcl‐2 levels, indicating a shift toward a pro‐apoptotic profile. This imbalance was accompanied by significantly increased cytochrome‐c protein levels, together with higher cleaved caspase‐9 and cleaved caspase‐3 protein levels. Collectively, these coordinated alterations in intrinsic apoptosis‐associated proteins support activation of apoptosis‐related signaling in MTX‐treated OVCAR‐3 cells.

The observed increase in cytochrome‐c protein levels together with elevated cleaved caspase‐9, apoptosome formation and activation of the initiator caspase, caspase‐9, represent hallmark steps of the intrinsic pathway [38, 39]. In line with this mechanism, MTX significantly increased cleaved caspase‐9 levels, followed by a marked elevation in cleaved caspase‐3 expression, consistent with coordinated alterations in intrinsic apoptosis‐associated protein markers. Thus, the present findings support involvement of mitochondrial intermediate signaling and initiator caspase activation at the protein level, extending beyond assessment of the Bax/Bcl‐2 ratio alone. The significant attenuation of cytochrome‐c protein levels and caspase‐9 activation by antioxidant agents further supports the concept that redox‐dependent mitochondrial apoptosis is amenable to modulation. The present investigation specifically focused on the intrinsic mitochondrial apoptotic pathway, which is principally regulated through increased cytochrome‐c protein levels and caspase‐9 activation. Caspase‐8, a key initiator of the extrinsic death receptor‐mediated pathway, was not evaluated in the current study. Therefore, potential interactions between intrinsic and extrinsic apoptotic signaling during MTX exposure remain to be clarified in future studies.

An important observation is that antioxidant co‐treatment did not completely abolish MTX‐induced apoptotic responses but significantly attenuated them. This finding suggests that these agents may not fully block cytotoxicity but rather recalibrate cellular stress responses by partially restoring redox balance. Among the concentrations examined, resveratrol produced the largest modulatory effect on both the Nrf2/HO‐1 axis and mitochondrial apoptotic signaling, supporting its multi‐target regulatory profile [21]. The differential responses observed among antioxidant compounds may reflect their distinct molecular targets and biological properties related to redox regulation, which could contribute to variations in their ability to modulate MTX‐induced cellular stress responses.

The MTT viability analysis provided additional functional support for the biochemical findings observed in the present study. MTX exposure significantly reduced OVCAR‐3 MTT metabolic activity, which was accompanied by increased oxidative stress, reduced Nrf2 and HO‐1 protein expression, and activation of apoptosis‐associated signaling markers. Antioxidant co‐treatment partially restored MTT metabolic activity, consistent with their ability to attenuate redox imbalance and modulate apoptosis‐related proteins. Among the concentrations examined, resveratrol produced the largest increase in MTT metabolic activity, paralleling its more pronounced effects on oxidative stress parameters and apoptosis‐associated signaling. These findings suggest that preservation of cellular viability may be closely associated with modulation of oxidative stress and mitochondrial apoptotic responses under MTX exposure.

Nevertheless, the observed restoration of MTT metabolic activity should be interpreted with caution. Although attenuation of oxidative stress and apoptosis may appear protective at the cellular level, methotrexate is an anticancer agent whose therapeutic efficacy is partly mediated through the induction of oxidative stress and apoptotic cell death. Therefore, antioxidant‐mediated suppression of these responses could theoretically reduce antitumor activity in ovarian cancer cells. The present study was designed to investigate redox‐associated and apoptosis‐related signaling mechanisms rather than therapeutic outcomes. Therefore, additional studies incorporating proliferation assays, clonogenic survival analyses, and tumor response models are required to determine whether antioxidant co‐treatment ultimately enhances or compromises the anticancer effects of MTX.

A major strength of this study lies in the integrated evaluation of oxidative stress parameters, redox regulatory proteins, and intrinsic apoptotic mediators within the same cellular model and time frame at the protein level. This approach provides a more comprehensive assessment of MTX‐induced cellular effects beyond descriptive oxidative stress changes and supports an association between alterations in redox homeostasis and apoptosis‐associated signaling.

Although oxidative stress was evaluated using TAC, TOC, OSI, and MDA measurements, intracellular ROS levels were not directly quantified. Direct ROS assessment using fluorescence‐based approaches such as DCFH‐DA staining would provide additional evidence regarding the magnitude of oxidative stress induced by MTX. Future studies incorporating direct ROS measurements may further clarify the relationship between redox imbalance and apoptotic signaling in ovarian cancer cells.

Beyond its antineoplastic efficacy, methotrexate is associated with clinically significant adverse effects involving the liver, kidney, gastrointestinal tract, bone marrow, and other tissues. Oxidative stress and mitochondrial dysfunction have been implicated as important contributors to MTX‐associated toxicity. Although the present study was conducted in a malignant ovarian cell model, the observed alterations in redox homeostasis and apoptosis‐associated signaling are consistent with findings previously reported in non‐malignant tissues exposed to MTX. Therefore, further investigation of oxidative stress‐related pathways may contribute to strategies aimed at minimizing treatment‐associated toxicity while maintaining therapeutic efficacy.

Nevertheless, certain limitations should be acknowledged. The findings are derived from a single ovarian serous carcinoma cell line (OVCAR‐3), which represents only one molecular subtype of ovarian cancer. Therefore, caution should be exercised when extrapolating the present results to other ovarian cancer subtypes. Validation in additional ovarian cancer models, including SKOV‐3 and CAOV‐3 cells, would strengthen the generalizability of the findings and help determine whether the observed redox‐ and apoptosis‐related responses are conserved across distinct molecular backgrounds.

Another limitation is the absence of a non‐malignant ovarian epithelial control. Consequently, it cannot be determined whether the observed alterations in oxidative stress parameters, Nrf2 and HO‐1 protein expression, apoptosis‐associated signaling markers, and MTT metabolic activity are specific to malignant ovarian cells or represent more general cellular stress responses. Future studies comparing ovarian cancer cell lines with normal ovarian epithelial models would provide valuable insight into the cellular specificity and biological significance of these findings.

In addition, although Nrf2 and HO‐1 protein levels were quantified, functional activation of the Nrf2 signaling pathway was not directly assessed. Measurements of Nrf2 nuclear translocation, ARE‐dependent transcriptional activity, and downstream target gene expression would provide a more comprehensive evaluation of pathway activity. Therefore, the present findings should be interpreted as changes in protein expression rather than definitive evidence of pathway inhibition.

Moreover, all protein measurements were performed using ELISA‐based assays. Although ELISA provides a sensitive quantitative assessment of protein abundance, it does not permit evaluation of protein molecular weight, subcellular localization, or cleavage patterns. Therefore, confirmation of the present findings using complementary techniques such as Western blot analysis would further strengthen confidence in the observed alterations in Nrf2, HO‐1, cytochrome‐c, and apoptosis‐associated proteins.

Furthermore, apoptosis was inferred from changes in apoptosis‐associated protein markers, including Bax, Bcl‐2, cytochrome‐c, cleaved caspase‐9, and cleaved caspase‐3. Direct assessment of apoptotic cell death using methods such as Annexin V/PI staining, TUNEL analysis, mitochondrial membrane potential assays, or flow cytometry was not performed. Therefore, the present findings should be interpreted as evidence of apoptosis‐related signaling alterations rather than direct quantification of apoptotic cell death.

Finally, the study reflects acute (24‐h) exposure conditions; chronic MTX treatment may induce distinct redox adaptation dynamics. Although all experiments were conducted using independent biological replicates and yielded consistent trends across treatment groups, the relatively limited number of biological replicates should be considered when interpreting the findings. Future studies with larger sample sizes would further strengthen the reproducibility and statistical robustness of the observed results.

Although individual aspects of MTX‐induced oxidative stress, antioxidant defense impairment, and apoptosis have been reported previously, the present study provides an integrated evaluation of oxidative stress parameters, Nrf2 and HO‐1 protein expression, apoptosis‐associated signaling markers, and MTT metabolic activity within the same ovarian cancer model. This approach allows a more comprehensive assessment of the relationships between redox imbalance, apoptosis‐related signaling, and cellular viability under MTX exposure and antioxidant co‐treatment.

Collectively, the present findings indicate that MTX induces redox imbalance–associated coordinated alterations in intrinsic apoptosis‐associated protein markers in OVCAR‐3 cells, and that this process can be partially modulated by selected antioxidant agents. Simultaneous evaluation of Nrf2 and HO‐1 protein expression together with the cytochrome‐c–caspase‐9–caspase‐3 signaling pathway provides supportive evidence regarding MTX‐induced cellular stress responses at the protein level.

5. Conclusion

This study demonstrates that MTX is associated with marked redox imbalance in OVCAR‐3 ovarian cancer cells, leading to reduced Nrf2 and HO‐1 protein expression together with coordinated alterations in intrinsic apoptosis‐associated protein markers. Reduced antioxidant capacity, increased lipid peroxidation, Bax/Bcl‐2 imbalance, increased cytochrome‐c protein levels, and activation of caspase‐9 and caspase‐3 collectively support coordinated alterations in redox‐ and intrinsic apoptosis‐associated protein responses at the protein level.

Antioxidant co‐treatment partially restored redox signaling and attenuated alterations in intrinsic apoptosis‐associated protein markers. Among the concentrations examined, resveratrol produced the largest modulatory effect. These findings provide supportive evidence that MTX‐induced cellular stress is associated with alterations in redox homeostasis and mitochondrial apoptosis‐related signaling.

Although limited to a single cell line and acute exposure conditions, the results highlight the importance of integrated protein‐level analysis for understanding redox‐mediated chemotherapeutic responses and warrant further investigation in broader experimental models.

Author Contributions

Oya Korkmaz conceived and designed the study, performed the experiments, analyzed and interpreted the data, and wrote and revised the manuscript. The author has read and approved the final version of the manuscript.

Funding

The author has nothing to report.

Ethics Statement

As this study was conducted solely on a commercially available ovarian cancer cell line (OVCAR‐3) and did not involve human or animal subjects, ethical approval was not required.

Conflicts of Interest

The author declares no conflicts of interest.

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.